Polygala tenuifolia-Acori tatarinowii herbal pair as an inspiration for substituted cinnamic α-asaronol esters: Design, synthesis, anticonvulsant activity, and inhibition of lactate dehydrogenase study

European Journal of Medicinal Chemistry 183 (2019) 1 11650

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Research paper

Polygala tenuifolia-Acori tatarinowii herbal pair as an inspiration for

substituted cinnamic -asaronol esters: Design, synthesis,

anticonvulsant activity, and inhibition of lactate dehydrogenase study

a

,

, b,

Yajun Bai ^{a 1}, Xirui He ^{a 1}, Yujun Bai ^a, Ying Sun ^a, Zefeng Zhao ^a, Xufei Chen ^a, Bin Li ^a,

Jing Xie ^a, Yang Li ^a, Pu Jia ^a, Xue Meng ^{a e}, Ye Zhao ^a, Yanrui Ding ^c, Chaoni Xiao ^a,

,

Shixiang Wang ^a, Jie Yu ^a, Sha Liao ^a, Yajun Zhang ^a, Zhiling Zhu ^f, Qiang Zhang ^a,

Yuhui Zhao ^a, Fanggang Qin ^a, Yi Zhang ^a, Xiaoyang Wei ^a, Min Zeng ^a, Jing Liang ^a,

Ye Cuan ^a, Guangzhi Shan ^f^***, Tai-Ping Fan ^{d **}, Biao Wu ^a, Xiaohui Zheng ^a

,

, *

^aNorthwest University, Xi'an, 710069, China

^bDepartment of Bioengineering, Zhuhai Campus of Zunyi Medical University, Zhuhai, 519041, China

^cSchool of Biotechnology, Jiangnan University, Wuxi, 214122, China

^dDepartment of Pharmacology, University of Cambridge, Cambridge, CB2 1PD, UK

^eInstitute of Traditional Chinese Medicine, Shaanxi Academy of Traditional Chinese Medicine, Xi'an, 710003, China

^fInstitute of Medicinal Biotechnology, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, 100050, China

a r t i c l e i n f o

a b s t r a c t

Article history:

Inspired by the traditional Chinese herbal pair of Polygala tenuifolia-Acori Tatarinowii for treating epi-

Received 11 June 2019

Received in revised form

11 August 2019

Accepted 27 August 2019

Available online 6 September 2019

lepsy, 33 novel substituted cinnamic a-asaronol esters and analogues were designed by Combination of

Traditional Chinese Medicine Molecular Chemistry (CTCMMC) strategy, synthesized and tested sys-

tematically not only for anticonvulsant activity in three mouse models but also for LDH inhibitory ac-

tivity. Thereinto, 68e70 and 75 displayed excellent and broad spectra of anticonvulsant activities with

modest ability in preventing neuropathic pain, as well as low neurotoxicity. The protective indices of

these four compounds compared favorably with stiripentol, lacosamide, carbamazepine and valproic

acid. 68e70 exhibited good LDH1 and LDH5 inhibitory activities with noncompetitive inhibition type,

and were more potent than stiripentol. Notably, 70, as a representative agent, was also shown as a

moderately positive allosteric modulator at human a1b2g2 GABA_Areceptors (EC₅₀46.3 7.3 mM). Thus,

68e70 were promising candidates for developing into anti-epileptic drugs, especially for treatment of

refractory epilepsies such as Dravet syndrome.

Keywords:

Combination of traditional Chinese

medicine molecular chemistry

Polygala tenuifolia-Acori tatarinowii herbal

pair

Anticonvulsant compounds

Lactate dehydrogenase inhibitor

1. Introduction

and about a third of them will go on to develop epilepsy [3]. Un-

fortunately, even with optimal anti-epileptic drug (AED) therapy,

Epilepsy is the third most common chronic brain disorder

characterized by recurrent spontaneous seizures due to hyperex-

citability and hypersynchrony of brain neurons [1,2]. Globally,

about 10% of people will have at least one seizure in their lifetime,

about one third of patients have poor seizure control and become

medically refractory [4]. Even worse, adverse effects and drug-

resistance have become leading causes of treatment failure with

current AEDs, which also increase the risk of premature death [5],

injury, psychosocial dysfunction, as well as lowering the quality of

life [6,7]. Therefore, continuing efforts are needed to develop more

effective pharmaceutical agents with less adverse effects.

The development of AEDs is closely based on the understanding

of the mechanism of epileptic ﬁring. For decades, almost all of

clinically used AEDs (ﬁrst-, second- and third-generation AEDs) act

on the underpinnings of neuronal ﬁring by targeting ion channels

* Corresponding author.

** Corresponding author.

*** Corresponding author.

E-mail addresses: shanguangzhi@imb.pumc.edu.cn (G. Shan), tpf1000@cam.ac.

uk (T.-P. Fan), zhengxh@nwu.edu.cn (X. Zheng).

1

These authors contributed equally to this work.

https://doi.org/10.1016/j.ejmech.2019.111650

2

Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

and/or neurotransmitter receptors on the neural cells [8]. However,

in recent ten years, a different view has emerged that neuron-

helper systems, such as astrocytes, the vasculature, the immune

system and metabolic pathways, play a far greater role than ﬁrst

thought in seizure control [1,9e11]. For example, in 2015, Sada and

his co-workers reported that seizures and epileptiform activity

were reduced by inhibition of the metabolic pathway via lactate

dehydrogenase (LDH), a component of the astrocyte-neuron lactate

shuttle [12]. Among 20 clinical ﬁrst-line antiepileptic drugs, only

stiripentol (1, Chart 1, STP) had a partial LDH inhibitory effect [12].

Although STP exerted antiepileptic effect by two other widely

recognized mechanisms, namely, inhibition of coadministered

AEDs metabolism [13] and allosteric modulating of GABA receptors

[14], in fact, its partial LDH inhibitory effects, just like that of

ketogenic diet [12], might be the key to the treatment of Dravet

syndrome, a kind of refractory epilepsy for children [15]. Thus,

compounds with multi-target antiepileptic activity, especially LDH

inhibitory activity, might be developed as a new generation of AEDs

for the treatment of some drug-resistant or refractory epilepsies.

In the past ﬁve years, our continuous study of traditional Chi-

nese medicine (TCM) antiepileptic herbal pair (Polygala ten-

uifoliaꢀAcori Tatarinowii or YuanzhiꢀShichangpu) and Acori

In the present study, 30 new substituted cinnamic esters of

asaronol, 3,4,5-TMCA ester of 3-(2,4,5-trimethoxyphenyl)propan-

1-ol (4, Fig. 1), 3,4,5-TMCA amide of

-3⁰-aminoasarone (5, Figs. 1)

and 3'-aminodihydroasarone (6, Fig. 1) were synthesized. Their

anticonvulsant activities, anti-neuropathic pain, neurotoxicity, LDH

inhibitory activity, and even ADME of selected compounds were

systematically studied to ascertain if some of them were potential

candidates for further development as new AEDs to treat refractory

epilepsy. Moreover, molecular docking studies were performed to

further rationalize the biological results.

a-

a

2. Design rationale

The strategy of CTCMMC is founded upon two aspects: clinical

effectiveness of TCM and combinatorial chemistry. Using this

strategy, biologically active lead structures or fragments, stemming

from different herbs in the ‘composite formulae’ (fufang), were

selected or optimized without losing their functionalities, and then

constructed into a set of brand-new molecules by chemosynthetic

means in the light of compatibility principle of TCM (the principle

of Jun-Chen-Zuo-Shi [28,29]). Ultimately, one or more potent mol-

ecule(s) were produced with comparable or better activity

compared with the original ‘composite formulae’. The goal of

CTCMMC is not only to rationalized and simplify the components of

TCM fufang, but also to fuse combination of active components by

chemical means aiming to optimize the efﬁcacy of such resultant

molecules.

Tatarinowii lead to ﬁnd a

-asaronol (3, (E)-3⁰-hydroxyasarone, Chart

1), which showed better LDH inhibition than STP (unpublished). In

addition, we found that 3 exhibited better general antiepileptic

activities in maximal electroshock seizure (MES), pentylenete-

trazole (PTZ) and 3-mercaptopropionic acid (3-MP)-induced mice

seizures, and possessed lower acute toxicity than carbamazepine

In this study, a-asaronol (3) and 3,4,5-TMCA (20) are the two

(CBZ), STP and

a

-asarone (2, Chart 1, one of key active components

complementary active components were selected from Acori

Tatarinowii and Polygala tenuifolia respectively to serve as the

parent compounds, which were then derived into two subsets of

libraries: section A and B compounds (Figs. 1 and 2). Like Polygala

tenuifolia-Acori Tatarinowii herb pair, the section A and B com-

pounds were simply conjugated by ester or amide bond to form

33 C ‘hybrid molecule’ (55e87), which were subjected to a series of

anticonvulsant evaluation to identify if some of these new com-

pounds exhibited more potent anticonvulsant activity than their

parent compounds or herbal pair.

of A. Tatarinowii) [16]. Metabolic studies had proved that 3 was a

metabolite of 2 [17]. In 2015, 2 was approved by China Food and

Drug Administration (CFDA) for the treatment of grand and petit

mal epilepsy. Nevertheless, three rare but potentially life-

threatening adverse effects of 2 (carcinogenic [17,18], teratogenic

[19,20] and genotoxic activity [21]) might restrict its clinical use. It

has been proved that these toxic effects of 2 mainly come from its

unstable epoxide metabolites (cis and/or trans-asaron-1⁰,2⁰-

epoxide) by forming DNA adducts, but not metabolite 3 [22]. In

addition, (E)-3-(3,4,5-trimethoxyphenyl)acrylic acid (3,4,5-TMCA,

20), one of active components of Yuanzhi also exhibited antiepi-

leptic effects by modulating GABA_Aergic systems [23]. Some of its

amide derivatives displayed good antiinﬂammatory and neuro-

protective actions [24,25]. Thus, taking inspiration from old anti-

epileptic herbal pair of YuanzhiꢀShichangpu and using our drug

design strategy (Combination of Traditional Chinese Medicine

Molecular Chemistry, CTCMMC) [26] combined with hybrid mole-

cules approach [27], we ﬁrstly designed a hybrid molecule 68

(Scheme 3). To our delight, 68 exhibited extraordinary antiepileptic

activity in MES test. Therefore, further study the structure-activity

relationship (SAR) of other analogues of 68 seems more

meaningful.

3. Results and discussion

3.1. Chemistry

Section A compounds 3e6 were synthesized in a serial of classic

reactions according to Scheme 1. Brieﬂy, treatment of Meldrum's

acid with methanol in toluene at reﬂux temperature followed by

addition of 2,4,5-trimethoxybenzaldehyde 37, pyridine and piper-

idine only afforded trans isomerism of cinnamic ester 38 (92.5%

yield) [30], which was conﬁrmed by H NMR analysis (see sup-

porting information). Initially, conventional lithium aluminum

hydride (LAH) selective reduction of the cinnamic ester 38 to the

alcohol 3 was investigated [31], but we found an inseparable

mixture of 3 and 4 (about 9:1 ratio based on HPLC analysis). 3,

however, by DIBAL-H [32] as reductant, was obtained in high yield

(91%) without producing any over-reduction product 4. Catalytic

hydrogenation of mixture of 3 and 4 or pure 3 almost quantitatively

led to pure 4. It was worth mentioning that direct amination of 3

with sodium azide and 2 equivalents of triphenylphosphine to form

5 by Reddy's one pot methodology [33] was proved fruitlessly.

Thus, Mitsunobu reaction condition [34,35] to give phthalimide 39

was followed by hydrazinolysis of the phthaloyl group [36] to afford

primary amine 5, which was subjected to palladium catalyzed hy-

drogenation condition to afford compound 6 in nearly quantitative

yield.

A

Chart 1. Compounds with subunit of propenylic phenylpropene.

Reagents and conditions: (a) Meldrum's acid, MeOH, toluene,

Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

3

Fig. 1. Source of design ideas and the general structures of compounds designed.

reﬂux, 5 h, then pyridine/piperidine, rt, 24 h; (b) DIBAL-H,

THF, ꢀ45 ^ꢁC ~ ꢀ60 ^ꢁC, 2 h; (c) Pd/C (5%, wet), H₂, rt, 20 h; (d) o-

phthalimide, P(Ph)₃, DIAD, THF, 0 ^ꢁC ~ rt, overnight; (e) EtOH,

NH₂NH₂$H₂O (80%), 76 ^ꢁC, 3 h.

Most of section B compounds could be obtained from com-

mercial sources except some muti-methoxy-substituted cinnamic

acids, such as 13, 17, 19 and 21e24. Thus, these unusual cinnamic

acids were synthesized in 2e10 steps using the following synthetic

procedures (Scheme 2). As above mentioned (Scheme 1, condition

(a)), the Meldrum's acid was treated with methanol to yield the

monomethyl malonate which, in the same pot, was treated with

aldehydes 40 in the presence of pyridine and piperidine to yield the

desired methyl cinnamates derivatives. Subsequent hydrolysis of

this methyl ester with aqueous NaOH/methanol solution furnished

13 in quantitative yield, and further puriﬁcation was not required.

The desired intermediates of 17, 19 and 21e24 were also prepared

in a similar manner from their corresponding aldehyde precursors.

The oleﬁn geometry of these cinnamic acid derivatives were

assigned as (E) in each case, based on the H NMR coupling constant

of the vinyl protons. (See supporting information).

Treatment of aldehyde 44 with m-CPBA under Baeyer-Villiger

oxidation conditions [37] gave 2,3,4-trimethoxyphenyl formate

which was hydrolyzed to give desired 2,3,4-trimethoxyphenol,

followed by subsequent methylation with dimethyl sulfate to

obtain the 1,2,3,4-tetramethoxybenzene 45 in 70.6% yield over

three steps. This procedure was also employed smoothly in the

synthesis of intermediates 47, 50 and 53.

For the synthesis of aldehyde intermediates 42, 46, 48, 51 and

54, four different synthetic procedures were tried and evaluated

(Schemes 2; conditions a, e-g), which were brieﬂy listed in

Supplemental Table 1. Initially, preparation of the aldehyde 51 was

accomplished in 87% yield by treatment of compound 50 with

POCl₃and DMF (Scheme 2, conditions (g)) [38]. However,

attempting to use this formylation method to prepare the aldehyde

46, 48 and 54 was unsuccessful. Thus, falling back on other classical

formylation methods, such as POCl₃/PhN(CH₃)CHO [39], TFA/HMTA

[40] and n-BuLi/DMF [41,42], we successively achieved the com-

pound 42, 46, 48 and 54 in moderate to good yield (42.9e89.4%).

The preparation of ﬁnal products 55e85 were achieved as

depicted in Scheme 3. Treatment of compound 4 with commer-

cially available compound 20 under standard Steglich esteriﬁcation

conditions afforded the 85 in 71% yield after chromatography [43].

This procedure was also employed smoothly in the synthesis of

Fig. 2. Section B compounds.

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Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

Scheme 1. Synthesis of intermediates of section A compounds (3e6)^a.

Scheme 2. Synthesis of intermediates of section B compounds (13, 17, 19 and 21e24)^a.

^aReagents and conditions: (a) THF, n-BuLi, DMF, ꢀ40 ^ꢁC ~ ꢀ60 ^ꢁC, 4 h; (b) m-CPBA, CH₂Cl₂, 0 ^ꢁC ~ rt, 24 h; (c) MeOH, NaOH, 0 ^ꢁC ~ rt, 2 h; (d) K₂CO₃, acetone, Me₂SO₄, reﬂux, 20 h; (e)

TFA, HMTA, 70 ^ꢁC, 8 h; (f) POCl₃, PhN(CH₃)CHO, rt, 18 h; (g) POCl₃, DCM, DMF, reﬂux, 4 h; (h) Meldrum's acid, MeOH, toluene, reﬂux, 5 h, then pyridine/piperidine, rt, 24 h.

other ﬁnal products 55 to 84 with yield of 57e91%. Thereinto, the

crystal structure of representative compounds 65 and 78 were

conﬁrmed by three-dimensional X-ray diffraction data (Fig. 3).

In addition, compounds 20 underwent standard EDCI-coupling

conditions with saturated or unsaturated 2,3,4-trimethoxy-

substituted amphetamine (5 or 6) to afford ﬁnal amides 86 and 87

in 80.8% and 86.6% yield, respectively (Scheme 4). Much effort was

not made to optimize the yields of these ﬁnal products.

and physical properties) of all the intermediates and ﬁnal products

in the Experimental Section. In the Supporting Information, we

provided full spectroscopic data for all synthetic compounds pre-

pared in this study.

3.2. Pharmacology

3.2.1. MES and neurotoxicity screening

We reported the details (synthetic procedure, characterization

Initially, we selected the mouse MES test as a basic evaluation of

Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

5

Scheme 3. Synthesis of section C compounds (55e85)^a.

a

Reagents and conditions: (a) EDCI, DMAP, 10e18 h, DCM, rt. (yield: 57e91%).

anticonvulsant activity, because it was the most widely accepted

seizure model remaining the ‘valued and gold standards’ in early

stages of discovery of new AEDs [44,45]. The neurotoxicity of all

ﬁnal products was also screened by the rotorod test [46] at this

stage. Table 1 presented the preliminary results of compounds

55e87 (dose of 100 mg/kg) at the MES test with ﬁve pretreatment

times (0.5, 1, 2, 3 and 4 h) and rotorod test with three pretreatment

times (0.5, 1 and 2 h). All compounds were administered intraper-

itoneally (ip) to mice, unless otherwise indicated. Surprisingly, our

data indicated that all compounds displayed potent anticonvulsant

activity and had no neurotoxicity within 2 h of drug administration

(Table 1). In the whole series, 29 compounds 55e71, 74e78, 80e82,

and 84e87 revealed rapid onset within 0.5 h, while 26 compounds

displayed long-lasting protection against convulsion showing ac-

tivity up to 2 h (56, 59, 62e66, 70, 77, and 86), 3 h (57, 58, 60, 61,

67e69, 74e76, 80e82, and 85) and even 4 h (55, 75), respectively.

Except for weak active molecules (72, 73, 79 and 83), all other

compounds showed 25e100% protection lasting from 0.5 h up to

4 h. Thereinto, 6 compounds (55, 64, 65, 68, 69, and 75) exhibited

most potent with maximal protection (100%) at 0.5 h, which clearly

increased over that of their parent sections (3 and 20) and standard

drug (STP). Slightly weaker efﬁcacy was observed at the same time

point, when R was 3-methoxyl (56), 3,4-dimethoxyl (62), 2,3,4,6-

tetramethoxyl (70), 2-chlorine (76), 3-bromine (80), and 2-

triﬂoromethyl (82). Additionally, we also systematically compared

the effect of methoxyl group in amounts and position as well as

ﬂuoro, chloro, bromo, and triﬂuoromethyl group placed at benzene

backbone of cinnamic acid (section B, Fig. 2) on MES activity. By and

large, most of compounds with methoxyl group were better than

those with halogen group in the MES test (Table 1). As to the

methoxylated compounds, the number and position of methoxyl

lead to different anti-MES activity, which might be associated with

the electron donor and/or the steric effect of methoxyl for phenyl

ring. As to the halogenated compounds, with the exclusion of

highly active compound 75, all exhibited low or modest MES ac-

tivity in mice. It was worth noting that the exchange of electron-

donating (ED) methoxyl in the ortho-position of the cinnamic

acid moiety with electron-withdrawing (EW) groups, such as ﬂuoro

(73), chloro (76), bromo (79), and triﬂuoromethyl (82), resulted in

the reduction of the MES activity. These observations suggested

6

Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

Fig. 3. Crystal structures and Oak Ridge Thermal-Ellipsoid Plot Program (ORTEP) drawing of 65 and 78, showing the atomic numbering. Thermal ellipsoids are drawn at 50%

probability.

Scheme 4. Synthesis of section C compounds (86 and 87)^a.

a

Reagents and conditions: (a) EDCI, HOBt, DCM, 10 h, rt. (yield:80e86%).

that other ED groups in position 2 might potentially improve

anticonvulsant properties. Finally, we brieﬂy examined the effect of

activity in section A. When R was 3,4,5-trimethoxyl, we observed

that 4, 5, and 6 moieties provided compounds (85, 86, and 87

respectively) with lower protection in the MES test compared to

compound 68. Therefore, the double bond and 3⁰-oxygen atom in

pretreatment times 0.5,1, 2, 3 and 4 h (Table 2). The results revealed

that 14 compounds (55, 57, 62, 64, 65, 67e71, 75, 76, 78, and 81)

showed modest to good activity (at least of 50% protection in the

MES test). Among these compounds, 55, 68e70, 75, and 76 showed

longer protection time starting from 0.5 h to 2 h with different

maximal protective effects, i.e., 75% protection for 55, 68e70, 75,

and 76 or 50% protection for 62, 64, and 65. While compounds 56,

66, 80, and 82 showed short duration of anti-MES activity with

weak or no protective effect at the same dose level.

the

a-asaronol might simultaneously play a key role in anticon-

vulsant properties.

Next, we further evaluated 18 active compounds from Table 1 in

the mouse-MES test (ip) at the dose of 50 mg/kg, at ﬁve

Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

7

Table 1

Anticonvulsant activity and acute neurotoxicity of compounds 55e87: MES and rotorod test in mice (ip^a, dose of 100 mg/kg).

MES^b

0.5 h

TOX^c

0.5 h

0

Compd

R

m

Sub. Site

X

C₇-C₈

1 h

2 h

3 h

4 h

1 h

2 h

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

3

OMe

F

Cl

Br

1

2

3

4

5

1

3

e

2

3

4

2, 3

2, 4

2, 5

2, 6

3, 4

3, 5

2, 3, 4

2, 3, 6

2, 4, 5

2, 4, 6

3, 4, 5

2, 3, 4, 5

2, 3, 4, 6

2, 3, 5, 6

2, 3, 4, 5, 6

2

3

4

2

3

4

2

3

4

2

3

4

O

N

e

d

s

4^d/4^e

3/4

2/4

3/4

2/4

4/4

2/4

4/4

3/4

2/4

1/4

2/4

4/4

3/4

2/4

1/4

3/4

2/4

3/4

1/4

2/4

3/4

4/4

3/4

2/4

1/4

2/4

3/4

2/4

4/4

3/4

1/4

2/4

4/4

2/4

1/4

0/4

3/4

2/4

1/4

3/4

2/4

3/4

2/4

1/4

2/4

1/4

2/4

1/4

2/4

0/4

1/4

3/4

1/4

0/4

1/4

3/4

2/4

1/4

0/4

1/4

0/4

2/4

1/4

0/4

1/4

0/4

1/4

0/4

1/4

2/4

1/4

0/4

1/4

2/4

1/4

0/4

1/4

0/4

1/4

0/4

1/4

0/4

1/4

0/4

0^f/4^e

0/4

CF₃

OMe

e

3, 4, 5

e

d

s

e

20

e

STP^g

e

a

The compounds (prepared in 0.5% Tween-80 and 0.9% normal saline) were administered intraperitoneally.

Maximal electroshock test (the animals were examined at ﬁve pretreatment times: 0.5, 1, 2, 3, and 4 h).

Neurological toxicity (determined from the rotarod test at three pretreatment times: 0.5, 1, and 2 h).

Data indicate number of mice protected.

number of mice tested.

the number of mice affected.

b

c

d

e

f

g

STP ¼ stiripentol.

3.2.2. ScPTZ-induced seizures test

be stressed, however, that compounds 69 and 70 exhibited both

rapid onset and long-lasting anti-PTZ protection showing activity

up to 2 h, whereas compound 62 showed shorter duration of action

(within 1 h).

In general, all of the test compounds were ineffective in scPTZ-

induced clonic seizures at the dose of 100 mg/kg and 300 mg/kg,

but exhibited satisfactory protection in scPTZ-induced tonic seizure

at the dose of 300 mg/kg (Table 3). Compounds 57, 62, 67, 69 and 75

were the most effective with 100% inhibition of tonic seizure.

Furthermore, compounds 56, 57, 62, 67, 69, 75, 76, 78, and 80e82

showed complete protection against scPTZ-induced mortality at

higher dose.

Additionally, compounds emerging in Table 2 were also evalu-

ated by scPTZ seizure threshold test in mice. The results combined

with corresponding clonic seizures rate (CSR), tonic seizure rate

(TSR) and mortality rate (MR) proﬁles were presented in Table 3. All

of the tested compounds exhibited satisfactory activity at dose of

300 mg/kg (at least of 50% protection), whereas compound 69e71

showed marginal protection at dose of 100 mg/kg (at most of 25%

protection), and other compounds were completely inactive at the

lower dosage. Thereinto, compound 57, 62, 67, 69, and 75 showed

relatively excellent protective effect at dose of 300 mg/kg. The

times to peak effect of these compounds were all at 0.5 h for 100%

protection, while the points of 75% protection were found at 0.25 h

and 1 h for 69, and at 1 h for 57, 62, and 75. Slightly weaker efﬁcacy

was observed for compounds 64, 68, 70, 71, 76, 78, 80, and 81 with

maximal peak effect for 75% protection during 0.25 he1 h. It should

3.2.3. Sc3-MP-induced seizures test

Further evaluations of above selected 18 compounds were un-

dertaken using the sc3-MP model, which was used to predict

8

Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

Table 2

Table 3

Anticonvulsant activity: MES test in mice (ip^a, dose of 50 mg/kg).

Anticonvulsant activity: scPTZ test in mice ip^a.

MES^b

Compd Dose (mg/kg) Pretreatment time^b

Pretreatment time

(0.5 h)^c

0

Compd

R

m

Sub. Site

X

C₇-C₈

0.5 h

1 h

2 h

3 h

4 h

0.25 h 0.5 h 1 h 2 h CSR % TSR % MR %

55

56

57

62

64

65

66

67

68

69

70

71

75

76

78

80

81

82

OMe

F

1

2

3

4

1

2

3

4

3, 4

2, 3, 4

2, 3, 6

2, 4, 5

2, 4, 6

3, 4, 5

2, 3, 4, 5

2, 3, 4, 6

2, 3, 5, 6

4

O

d

3^c/4^d2/4 1/4 0/4 0/4

1/4

2/4

1/4

2/4

3/4

2/4

3/4

2/4

1/4

2/4

0/4

0/4 0/4 0/4 0/4

2/4 0/4 0/4 0/4

2/4 1/4 0/4 0/4

1/4 1/4 0/4 0/4

0/4 0/4 0/4 0/4

2/4 1/4 0/4 0/4

1/4 0/4 0/4 0/4

1/4 1/4 0/4 0/4

2/4 1/4 0/4 0/4

2/4 0/4 0/4 0/4

1/4 1/4 0/4 0/4

1/4 0/4 0/4 0/4

0/4 0/4 0/4 0/4

55

56

57

62

64

65

66

67

68

69

70

71

75

76

78

80

81

82

300

100

300

100

300

100

300

100

300

100

300

100

300

100

300

100

300

100

300

100

300

100

300

100

300

100

300

100

300

100

300

100

300

100

300

100

1^d/4^e

0/4

1/4

0/4

2/4

0/4

2/4

0/4

2/4

0/4

2/4

0/4

2/4

0/4

2/4

0/4

2/4

0/4

3/4

0/4

3/4

0/4

2/4

1/4

2/4

0/4

2/4

0/4

2/4

0/4

2/4

0/4

2/4

0/4

2/4

0/4

2/4

0/4

2/4

0/4

4/4

0/4

4/4

0/4

3/4

0/4

2/4

0/4

2/4

0/4

4/4

0/4

3/4

0/4

4/4

1/4

3/4

1/4

3/4

1/4

4/4

0/4

3/4

0/4

3/4

0/4

3/4

0/4

3/4

0/4

2/4

0/4

2/4 0/4 100

0/4 0/4 100

1/4 0/4 100

0/4 0/4 100

3/4 1/4 100

0/4 0/4 100

3/4 0/4 100

0/4 0/4 100

3/4 1/4 100

0/4 0/4 100

2/4 0/4 100

0/4 0/4 100

1/4 0/4 100

0/4 0/4 100

2/4 0/4 100

0/4 0/4 100

2/4 1/4 100

0/4 0/4 100

3/4 2/4 100

50

100

50

100

0

100

0

100

25

100

50

100

25

100

0

100

25

100

0

75

25

75

0

25

50

0

75

0

50

0

50

25

75

50

75

25

75

0

50

25

75

0

25

50

0

75

0

50

0

75

0

75

0

Cl

Br

2

4

3

4

CF₃

2

a

The compounds (prepared in 0.5% Tween-80 and 0.9% normal saline) were

administered intraperitoneally.

14

0/4 100

b

Maximal electroshock test (the animals were examined at ﬁve pretreatment

3/4 2/4 100

1/4 0/4 100

2/4 2/4 100

1/4 0/4 100

3/4 1/4 100

0/4 0/4 100

2/4 1/4 100

0/4 0/4 100

2/4 1/4 100

0/4 0/4 100

2/4 0/4 100

0/4 0/4 100

2/4 1/4 100

0/4 0/4 100

2/4 0/4 100

0/4 0/4 100

times: 0.5, 1, 2, 3, and 4 h).

c

Data indicate number of mice protected.

The total number of mice tested.

d

100

25

100

25

100

25

100

25

potential GABAergic efﬁcacy of new drugs [47]. The results

combining with three closely related protective parameters (CSR,

TSR and MR) were simultaneously listed in Table 4. In general,

compounds 68e70, 76, 78, and 80 displayed modest activity with

short durations (0.25 he1 h). The times to peak effect of these

compounds were all at 0.5 h for 50% protection. Notably, compound

55, in spite of exhibiting excellent proﬁle in the MES model

(Tables 1 and 2), together with 57, and 66 showed complete inac-

tivity in this test (Table 4), while others only showed marginal ac-

tivity during the same period.

Furthermore, consistent with the results in the scPTZ model,

none provided protection of clonic seizures at doses of 300 mg/kg.

Additionally, only 69, 70, 76, 78, and 80 showed modest protection

of tonic seizure. However, it appears that except compounds 55, 57,

81, and 82, all other compounds were found to be effective in sc3-

MP-induced mortality test with at least 75% protection of mice.

100

50

100

25

0

75

a

The selected compounds (prepared in 0.5% Tween-80 in 0.9% normal saline)

were administered intraperitoneally.

Subcutaneous injection PTZ (85 mg/kg, prepared in 0.9% normal saline) at four

pretreatment times after drug administration: 0.25, 0.5, 1, and 2 h.

The CSR (clonic seizures rate), TSR (tonic seizure rate) and MR (mortality rate)

induced by subcutaneous injection PTZ at pretreatment times 0.5 h.

b

c

d

Numbers of mice protected.

Total numbers of mice tested.

e

ED₅₀¼ 70.6 mg/kg, 183.6

mmol/kg) at the cinnamic phenyl moiety,

3.2.4. Quantitative evaluation against MES-, scPTZ-, and sc3-MP-

induced seizures

the anticonvulsant activity decreased, suggesting that methoxyl at

C-2 position was favored. We also found that one of the principal

active parts resided in 3, 4-dimethoxyl phenyl substructure

(R ¼ OMe, m ¼ 2) of cinnamate moiety as seen with compound 62

The excellent potencies of some selected compounds (55, 62, 64,

65, 68e71, and 75) in aforementioned anticonvulsant activity tests

(Tables 1e4) led us to further investigation and quantiﬁcation of

their pharmacological properties in mouse-MES, -PTZ, -3-MP, -Tox,

and -formalin tests. The formalin test was used to determine

whether active compounds could relieve the neuropathic pain [48],

which was one of the most insufferable symptom accompanying

seizure ﬁring [49]. Thus, the results of the median effective doses

(MES ED₅₀¼ 58.1 mg/kg, 140.2

mmol/kg), while other dimethoxyl

positional isomers, including 58, 59, 60, 61, and 63, showed similar

but lower protection in the MES test (Table 1). With these two

points in mind, we were not surprised to ﬁnd that 2,3,4-

trimethoxyl (64, ED₅₀¼ 33.4 mg/kg, 75.1

trimethoxyl (68, ED₅₀¼ 33.2 mg/kg, 74.7

tetramethoxyl (69, ED₅₀¼ 30.9 mg/kg, 65.1

tetramethoxyl (70, ED₅₀¼ 33.9 mg/kg, 71.4

provided excellent anticonvulsant activity in the MES test, while

mol/kg)

mmol/kg), 3,4,5-

m

mol/kg), 2,3,4,5-

(ED₅₀) and median toxic dose (neurological impairing dose, TD₅₀

)

mmol/kg) and 2,3,4,6-

combined with quantitative (dose range) testing in mice (ip) were

summarized in Table 5. These values were calculated from dose-

response curves containing 4e6 dose points (n ¼ 8e10 per dose).

Moreover, the protective indices (PI ¼ TD₅₀/ED₅₀), which were the

measure of the beneﬁt-to-risk ratio for selected compounds, and

partial protective proﬁles of four clinical AEDs: lacosamide, valproic

acid, stiripentol, and carbamazepine were also listed in Table 5.

Analysis of these data revealed that when the position of

mmol/kg) analogues

2,3,5,6-tetramethoxyl (71, ED₅₀¼ 57.9 mg/kg, 122.0

m

showed dramatically lower activity proﬁle. However, this trend was

not applicable for 2,3,4,5,6-pentmethoxyl analog (72) which gave

almost no activity in the MES test (Table 1). The reason for such

discrepancy is unclear but is under investigation. Additionally, our

data in MES test indicated that the electronic and hydrophobic

properties of phenyl substituents might impact on compounds’

methoxyl shifted from C-2 (55, ED₅₀¼ 39.0 mg/kg, 101.4

mmol/kg)

to C-3 (56, ED₅₀¼ 72.9 mg/kg, 189.6 mol/kg), or to C-4 (57,

m

Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

9

Table 4

to screen these novel

test.

a

-asaronol derivatives in the LDH inhibition

Anticonvulsant activity: sc3-MP test in mice (ip^a, dose of 300 mg/kg).

Pretreatment time^b

Pretreatment time (0.5 h)^c

The LDH inhibitory activities of these C series compounds were

measured by standard enzyme kinetic experiments on human

LDH1 and LDH5 puriﬁed isoforms [56]. Enzyme activity was

determined by the measurement of the absorbance decrease at

various wavelength (Supplemental Table 4), because the optimum

detection wavelength of NADH shifted under different solution of

synthetic compounds. Fig. 4 showed the inhibitory results of all the

synthesized compounds against LDH1 and LDH5. Thereinto, STP

served as LDH positive inhibitor while carbamazepine and valproic

acid served as negative inhibitor [12]. However, unlike reported

results (STP, 0.5 mM with ~30% inhibition ratio) [12], STP at the

same dose level showed no activity against both LDH1 and LDH5 in

our enzyme catalytic system (Fig. 4, STP₁). When the concentration

of STP was increased to 2.0 mM, a smaller boost of LDH1 and LDH5

inhibition ratio (7e10%) was observed (Fig. 4, STP₂). The inconsis-

tency between our test and literature value might be attributable to

the different LDH speciﬁc activity. Compound 60 and 66 were not

tested in this test because of their similar absorption spectrum with

NADH (Fig. S140, Supporting Information), which seriously inter-

fered the experimental results. With the exception of 62, none of

compd

0.25 h

0.5 h

1 h

2 h

CSR %

TSR %

MR %

55

56

57

62

64

65

66

67

68

69

70

71

75

76

78

80

81

82

0^d/4^e

1/4

0/4

1/4

0/4

1/4

0/4

1/4

0/4

1/4

0/4

1/4

0/4

1/4

2/4

1/4

2/4

1/4

0/4

1/4

0/4

1/4

0/4

1/4

0/4

1/4

0/4

100

75

100

75

50

75

50

75

50

25

50

25

0

25

0

50

a

The selected compounds (prepared in 0.5% Tween-80 and 0.9% normal saline)

were administered intraperitoneally.

Subcutaneous injection 3-MP (60 mg/kg, prepared in 0.9% normal saline) at four

pretreatment times after drug administration: 0.25, 0.5, 1, and 2 h.

The CSR (clonic seizures rate), TSR (tonic seizure rate) and MR (mortality rate)

induced by subcutaneous injection 3-MP at pretreatment times 0.5 h.

b

monomethoxyl-substituted or bismethoxyl-substituted a-asaronol

c

cinnamate (55e59, 61, and 63) presented in the Fig. 4 showed

inhibitory activities against LDH1 and LDH5. And 62 just exhibited

marginal inhibitory activity on LDH1, while almost inactivity on

LDH5 compared to the STP₂. Trimethoxyl-substituted and

d

Numbers of mice protected.

Total numbers of mice tested.

e

tetramethoxyl-substituted

a-asaronol cinnamates (64, 65, and

68e71) showed moderate inhibitory activity (8%~27% inhibition)

both on LDH1 and LDH5 without marked selectivity. Notably, the

2,4,6-trimethoxyl-substituted derivative 67 was originally shown

to be the most effective for inhibition of LDH1 (33% inhibition) and

LDH5 (26% inhibition) by UV-VIS spectrophotometry method

(Fig. 4), but later it was quickly revised to have a weak inhibition of

LDH (~5% inhibition) by mass method (Fig. 5). We thought that the

higher molar absorptivity (ε) of 67 might interfere the accuracy of

detection in the UV-VIS mode (Fig. S140, Supporting Information).

In comparison, compounds 64 and 68e70 exhibited good correla-

tions between LDH inhibition (~7%, 10%, 18%, 21% for LDH1 and ~8%,

13%, 15%, 27% for LDH5 respectively) and in vivo efﬁcacy (MES

anticonvulsant activity. Electron-donating groups with negative

Hammett s_pvalue (Supplemental Table 2.) maintained seizure

protection, while electron-withdrawing groups with positive

Hammett s_pvalue at the same positon decreased activity. By and

large, the higher value of s_pand/or

p was, the lower pharmaco-

logical activity it exhibited. All in all, 55, 64, 68e70, and 75 were the

most active compounds (ED₅₀: 30e40 mg/kg) in the MES test. Of

these compounds, 69 was observed to be the most active (MES

ED₅₀¼ 30.9 mg/kg, 65.1

times more potent than valproic acid, stiripentol, and carbamaze-

pine respectively (utilizing ED₅₀as mol/kg). Despite compound 69

mmol/kg), and it was 23.10, 3.96, and 1.17

m

showed lower protection (57.5% potency) in the MES test compared

to the lacosamide, its higher safety proﬁle in the rotorod test

(TD₅₀> 1000 mg/kg) resulted in 9.0-fold (MES), 64.0-fold (scPTZ),

and 18.2-fold (formalin) more beneﬁcial PI values than that of

lacosamide.

Additionally, three tetramethoxyl-substituted compounds

69e71 and two chloro-substituted compounds 76 and 78 showed

good activities in scPTZ test (ED₅₀(mg/kg), [mmol/kg]: 69, 155.3,

327.3; 70, 154.5, 325.6; 71, 149.2, 314.4; 75, 43.4, 116.5). Compounds

68e70 exhibited the most but moderate active in sc3-MP test (ED₅₀

(mg/kg), [mmol/kg]: 68, >200, >450.0; 69, >200, >421.5; 70, 202.9,

427.6), whereas halogen-substituted compounds (73e84) lacked

the activity in this test. In the formalin test, 69e71 and 75 displayed

good activity (ED₅₀(mg/kg), [mmol/kg]: 69, 42.2, 89.0; 70, 46.3,

ED₅₀¼ 75.1, 74.7, 65.1 and 71.4

mmol/kg respectively). Furthermore,

the LDH inhibitory activity of 68e70 were at least 4 times more

potent than STP. This unique pharmacological proﬁle of 68e70 in

LDH inhibitory test suggested they could serve as potential candi-

dates for treatment of refractory epilepsy such as Dravet syndrome,

in analogy to STP [57]. Compound 65 and 71 possessed moderate

LDH inhibitory activity (Fig. 4), while exhibiting lower in vivo efﬁ-

cacy (Table 5) comparing to 68e70. These relatively lower in vivo

activity exhibited by 65 and 71 could be due to a number of factors

that included oral bioavailability, biodistribution, and/or metabolic

stability. Curiously, pentamethoxyl-substituted

mate (72) was inactive at 0.5 mM in the LDH1/LDH5 inhibitory

model test. A similar trend with respect to halogen substituted

a-asaronol cinna-

a-

asaronol cinnamate (73e84) and compounds 85e87 were also

observed for the mean of LDH1/LDH5 inhibitory ratio < 5% and 9%

respectively. Perhaps it was because the space steric effect or cloud

density of molecule prevented binding with the LDH. In general, the

LDH inhibitory proﬁle of C series compounds, by and large, agreed

with the above animal model results.

97.6; 71, 50.8, 107.1; 75, 43.4, 116.5). Moreover, in accordance with

the results in Table 1, all selected compounds showed low neuro-

toxicity with TD₅₀value ~1000 or above (Table 5). In summary, it

appeared that compounds 68e70 and 75 emerged as promising

candidates with signiﬁcant and broad spectrum of anticonvulsant

activity.

Although, evaluation of LDH activity by UV-VIS spectropho-

tometry method typically had superior simplicity and sensitivity,

some substances, due to their having similar absorption wave-

length with NADH^þ, could seriously interfere the detection results.

To avoid this kind of interference, we employed another more

direct and sensitive method, LC-MS/MS (also termed as multiple

3.2.5. Lactate dehydrogenase inhibition

Sada et al. showed that seizures might be controlled by inhib-

iting LDH of nervous system [12]. As mentioned above,

a-asaronol

(3) had a good LDH inhibitory activity. Thus, it was highly necessary

10

Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

Table 5

Quantitative anticonvulsant data in mice (ip^a, ED₅₀, TD₅₀, and PI values).

Compd TPE

(h)^b

MES,^cED₅₀(mg/kg)

mol/kg]

scPTZ,^dED₅₀(mg/kg)

mol/kg]

sc3-MP,^eED₅₀(mg/kg) Formalin, ED₅₀(mg/kg) Tox,^fTD₅₀(mg/kg)

mol/kg] mol/kg] mol/kg]

PI^g

[m

MES PTZ 3-

Formalin

MP

55

0.5

39.0 (18.4e62.4)

[101.4]

72.9 (45.4e98.5)

[189.6]

70.6 (30.6e91.6)

[183.6]

58.1 (23.6e90.5)

[140.2]

33.4 (15.5e54.3)

[75.1]

53.5 (15.0e76.6)

[120.4]

73.5 (34.5e95.6)

[165.4]

69.8 (31.0e98.9)

[157.0]

33.2 (12.9e56.0)

[74.7]

30.9 (10.1e52.3)

[65.1]

33.9 (14.3e72.5)

[71.4]

57.9 (25.0e92.4)

[122.0]

34.6 (17.3e54.4)

[92.9]

58.7 (50.6e92.3)

[151.0]

53.4 (46.1e91.3)

[137.3]

75.7 (35.1e98.7)

[174.7]

240.3 (163.5e309.2)

[625.1]

247.1 (168.4e300.1)

[642.8]

243.9 (163.6e285.02) >300 [> 780.4]

[634.4]

173.2 (128.5e274.1)

[417.9]

202.8 (122.6e292.8)

[456.3]

211.7 (133.7e274.9)

[476.3]

>200; <300 [> 450.0; >300 [> 674.9]

<674.9]

211.7 (133.7e304.9)

[476.3]

203.8 (138.4e301.2)

[458.5]

155.3 (80.1e262.2)

[327.3]

154.5 (87.2e256.2)

[325.6]

149.2 (85.1e234.2)

[314.4]

167.2 (83.5e178.2)

[449.0]

149.8 (45.4e298.1)

[385.2]

141.7 (37.3e224.4)

[364.4]

>300 [780.4]

112.9 (79.9e156.5)

[293.7]

>100 [> 260.1]

>1000 [2601.3]

>1000 [> 2601.3]

>25.6 >4.2 z3.3 >8.9

>13.7 >4.1 >4.0 >8.9

>14.2 >4.1 z3.3 >8.2

>17.2 >5.8 z3.3 >10.7

>29.9 >4.9 z3.3 >12.8

>18.7 4.72 z3.3 z12.5

>13.6 z5 z3.3 z12.5

>14.3 >4.7 z3.3 >10.1

29.80 4.9 z3.3 11.6

>32.4 >6.4 >5.3 23.7

27.7 6.1 4.6 21.6

>17.3 6.7 z5 19.7

>28.9 >5.9 z5 23.0

>17.0 >6.7 >4.0 >9.9

>18.7 >7.1 >6.2 >9.3

>13.2 z5 >4.6 >8.9

56

248.8 (170.4e325.9)

[647.2]

57

121.8 (64.7e191.1)

316.8]

93.3 (85.4e211.7) [225.1] >1000 [> 2412.8]

78.4 (54.5e98.1) [176.4] >1000 [> 2249.8]

62

>300 [> 723.9]

>300 [> 674.9]

64

65

>80.0, <100.0 [> 180.0, >1000 [> 2249.8]

<225.0]

>80.0, <100.0 [> 180.0, >1000 [> 2249.8]

<225.0]

66

67

>300 [> 674.9]

>200 [> 450.0]

>200 [> 421.5]

99.0 (61.0e144.4) [222.7] >1000 [> 2249.8]

68

85.5 (49.0e109.0) [192.4] 988.1 (895.3e1090.6)

[2223.0]

42.2 (21.8e94.0) [89.0] >1000 [> 2107.4]

69

70

202.9 (146.7e305.0)

[427.6]

>300 [> 632.2]

46.3 (28.2e106.2) [97.6] 936.5 (850.3e1031.5)

[1973.6]

50.8 (31.8e109.0) [107.1] >1000 [> 2107.4]

71

75

>300 [> 805.6]

43.4 (30.2e104.7) [116.5] >1000 [> 2685.4]

76

248.3 (170.4e295.9)

[638.6]

>200 [> 514.4]

101.1 (45.0e145.9)

[260.0]

107.9 (71.4e157.3)

[277.5]

112.8 (76.8e157.6)

[260.3]

105.3 (62.4e153.0)

[243.0]

>1000 [> 2571.8]

>1000 [> 2307.9]

78

80

>200 [> 461.6]

215.4 (166.1e295.0)

[497.1]

>300 [> 692.4]

81

61.8 (25.7e95.0)

[142.6]

193.2 (114.4e274.5)

[445.9]

>200 [> 473.5]

1059.0 (864.6e1270.4) 17.1 5.5 z5 10.1

[2444.0]

>1000 [> 2367.4]

82

85.7 (50.5e98.8)

[202.9]

>300 [> 710.2]

122.9 (71.4e178.3)

[291.0]

>11.7 z5 z3.3 >8.1

LCM

VPAⁱ

STP^j

CBZ^k

3

9.4 ^h(8.1e10.7) [37.6] >500 ^h[> 1997.7]

15 ⁿ[0.1]

33.7 ^h(28.8e38.7)

[134.6]

372.9 (356.0e389.8)

[2585.8]

161.7 (146.2e256.2)

[690.1]

71.6 (45.9e135) [303.0] 3.96 <0.7

3.6

1.7

<0.1

2.2

216.9 (207.5e226.3) 239.4 (209.2.274.1)

1.6

[1504]

277 12.1 [1182]

[1660.1]

l

115 (99.14e133.39)

0.58 1.41

[490]^m

18.07 (13.66e34.88) >100 [> 423.2]

[76.5]

62.02 (41.72e87.6)

[276.6]

689.34 (556.53e818.17) 11.1

[3073.8]

a

The compounds were administered intraperitoneally to adult male KM mice. ED₅₀and TD₅₀values are in mg/kg (n ¼ 6). Numbers in parentheses are 95% conﬁdence

intervals determined by Probit analysis. A dash indicates not tested.

b

TPE: time of peak effect.

c

MES ¼ maximal electroshock seizure test.

d

PTZ ¼ pentylenetetrazole seizure test.

e

3-MP ¼ 3-mercaptopropionic acid.

f

Tox ¼ neurological toxicity. TD₅₀value determined from the rotorod test.

g

PI ¼ protective indices (TD₅₀/ED₅₀).

h

LCM ¼ lacosamide, reference [50].

i

VPA ¼ valproic acid, reference [51].

j

STP ¼ stiripentol.

k

CBZ ¼ carbamazepine, reference [52].

l

reference [53].

m

reference [54].

reference [55].

n

reaction monitoring (MRM)), to evaluate some representative

compounds (55, 67e70, and 75) and reference drug (STP) for LDH

inhibitory activity. The main advantage of MRM was the elimina-

tion of the effects of some uncaring substances in a complex

mixture [58]. In this test, the conversion of pyruvate to lactate was

selected again to evaluate the effect of LDH inhibition by moni-

toring the ion transition for lactic acid (m/z 89.0 / m/z 43.0) under

negative ion mode (Fig. S138, Supporting Information). The limit of

detection (S/N ¼ 5) and lower limit of quantiﬁcation (S/N ¼ 10) for

lactic acid were 0.25 and 0.5 ng/mL (Fig. S139, Supporting Infor-

mation), respectively, showing the higher sensitivity of the MRM

method. As shown in Fig. 5, the results of LDH1 and LDH5 inhibition

among compounds 68e70 were consistent with the pattern

observed from UV-VIS spectrophotometry method (Fig. 4).

Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

11

Fig. 4. Inhibition of human LDH1 and LDH5 activity by selected C series compounds (0.5 mM), measured by enzyme kinetic experiments. Values are reported as the mean (n ¼ 2 to 6

experiments in each compound.) with SEM. LDH activity in the direction of pyruvate-to-lactate were measured by using 1.0 mM pyruvate. The positive control agent (STP) were

detected in two concentrations (STP₁: 0.5 mM, STP₂: 2.0 mM) and the active level (~90%) of STP₂is indicated as a horizontal dotted line (black), while the two negative control agents

(CBZ and VPA: 0.5 mM) active level (~100%) is indicated as a horizontal dotted line (red). (For interpretation of the references to color in this ﬁgure legend, the reader is referred to

the Web version of this article.)

Fig. 5. Inhibition of human LDH1 (A) and LDH5 (B) activity by compound 67e70 (0.5 mM) and STP (2.0 mM), measured by MRM method. Values are reported as the mean (n ¼ 4 to 6

experiments in each compound.) with SEM. LDH activity in the direction of pyruvate-to-lactate were measured by using 1.0 mM pyruvate. The positive control agent (STP) is

indicated as a horizontal dotted line (black), while the blank control (without adding LDH) is indicated as a horizontal dotted line (red). (For interpretation of the references to color

in this ﬁgure legend, the reader is referred to the Web version of this article.)

However, compound 67, in MRM test, exhibited only weak inhibi-

tory activity for both LDH1 and LDH5, which agreed with its in vivo

moderate effect of the MES test (Tables 1 and 5). Additionally,

compounds 55 and 75 showed ~2% and ~5% inhibition for LDH1 and

LDH5 respectively (Fig. 5), which were consistent with the results

depicted in Fig. 4. In this regard, MRM method for detecting LDH

inhibition was more objective and credible than that of spectro-

photometry method in some cases.

verify their types of inhibition versus pyruvate. From secondary and

Lineweaver-Burk plots (Fig. 6), compounds 65, 68 and 69 were all

found to be noncompetitive inhibitors of LDH1 and LDH5 in the

conversion of pyruvate to lactate catalyzed by these two enzymes,

which suggested that these kind of inhibitors bound to the LDHs at

the different sites with pyruvate. In addition, their K_ivalues were

also determined from Lineweaver-Burk plots and reported in

Table 6.

On the basis of good LDH inhibitory activities of tri- and

tetramethoxyl-substituted

68 and 69 were selected for a complete enzyme kinetic analysis to

a-asaronol cinnamate, compounds 65,

3.2.6. GABA_Areceptor modulating activity

The g-aminobutyric acid receptor type A (GABA_Areceptor) is a

12

Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

Fig. 6. Secondary plots (SP) and Lineweaver-Burk plots (LBP) of the effects of representative compounds (65, 68 and 69) of two human LDHs (LDH1 and LDH5) activities. (A)

compound 65, (a) for LDH1, SP; (b) for LDH1, LBP; (c) for LDH5, SP; (d) for LDH5, LBP. (B) compound 68, (e) for LDH1, SP; (f) for LDH1, LBP; (g) for LDH5, SP; (h) for LDH5, LBP. (C)

compound 69, (i) for LDH1, SP; (j) for LDH1, LBP; (k) for LDH5, SP; (l) for LDH5, LBP. Control: enzyme activity without inhibitor. Inhibitors (65, 68 and 69) concentration: 1.0 mM.

These plots indicate non-competitive inhibition for both human LDH1 and LDH5. The data in each point were obtained from n ¼ 3e6. Data are mean SEM.

Table 6

postulated to play important roles in the etiology of epilepsy.

Among all known subtypes of the GABA_Areceptor, the a1b2g2 is

LDH1 and LDH5 inhibition constant (K_i) of compounds 65, 68 and 69^a.

the most abundant subtype that accounts for about 43% [62] and is

localized in many regions of the brain. It was therefore important to

Entry

Comd

LDH1 (K_i,

m

M)

LDH5 (K_i, mM)

1

2

3

65

68

69

11.98 1.21

14.11 1.52

1.77 0.16

3.52 0.24

3.35 0.31

4.84 0.51

evaluate effects of representative compound 70 on the

a1b2g2

receptor in the human HEK-293T cell line. HEK-293T cells in culture

a

for 12e24 h were studied with whole-cell patch-clamp recording

K_ivalues for each single inhibitor were determined using Lineweaver-Burk

plots. Values are reported as the mean (n ¼ 3 to 6 experiments in each com-

pound.) with SD. 100 mM sodium phosphate buffer (pH 7.5), 0.2 mM NADH, and

0.1 mMe1 mM sodium pyruvate.

technique. Application of 3.3e300

70 evoked GABA_Acurrents at 3

enhanced the response of 2 receptors to 3

average EC₅₀of 46.0 9.1 M and maximum potentiation of

310.5 22.5% (n ¼ 4) (Fig. 7A and B). Compound 70 was also a very

potent modulator with similar average EC₅₀of 46.3 7.3

m

M of stiripentol and compound

M (Fig. 7). Stiripentol strongly

M GABA, with an

m

a

1b

2g

m

ligand-gated chloride channel associated with a spectrum of re-

fractory seizures types, such as febrile seizures (FS), childhood

absence epilepsy (CAE) and Dravet syndrome etc [59e61]. A

number of antiepileptic drugs have agonistic effects on GABA_Are-

ceptors. Hence, modulation of GABA_Areceptors have been

mM (n ¼ 4)

(Fig. 7C and D). Notably, compound 70 had higher efﬁcacy than

stiripentol, with an average maximum potentiation of

355.7 20.4% (n ¼ 4).

Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

13

Fig. 7. Stiripentol (STP) and compound 70 increases the response to GABA in a concentration-dependent manner. HEK-293T cells were transfected with the a1 subunit along with b2

and

g

2 subunits. The peak current response to 11 s applications of GABA alone or with stiripentol and compound 70 respectively were measured in cells voltage-clamped at ꢀ50

mV. The GABA concentration used represented EC₁₀~EC₂₀for

a

1b2

g

2 isoform. (A) and (C) Representative traces in response to GABA alone and GABA (3

mM) with increasing

concentrations of stiripentol and compound 70 from 3.3 to 300

mM respectively. The amplitude of the current were increased by stiripentol and compound 70 in a concentration-

dependent manner. Concentration-response relationships were constructed by measuring the peak current with concentration of stiripentol (B) and 70 (D) respectively. Symbols

and bars represent the mean SEM. The EC₅₀of stiripentol for potentiation of the

a

1b

2g

2 isoform was 46.0 9.1

m

M (n ¼ 4), while that for compound 70 was 46.3 7.3 M (n ¼ 4).

m

3.3. Pharmacokinetics

exhibited rapid absorption (T_max¼ 35.56 5.5 min), high C_max

(1.89 0.58 g/mL), and AUC (138.4 10.4 mg∙min/L) as well as

m

Following in vitro studies, two compounds (69 and 70) were

selected for further studies. Their PK data (rats) were summarized

in Table 7. With a 20 mg/kg intravenous (iv.) dose, 69 and 70

formulated in 2.5% poloxamer physiological saline displayed AUC of

176.3 16.2 and 180.2 18.2 mg∙min/L in rats respectively. They

possessed high in vivo clearance (0.16 0.01 L/min/kg for 69 and

0.14 0.006 L/min/kg for 70), middle and low volume of distribu-

tion (1.98 0.28 L/kg for 69 and 2.14 0.28 L/kg for 70), and short

half-life (t_1/2¼ 19.72 4.2 min for 69 and t_1/2¼ 22.80 3.2 min for

70) in rats. In addition, compound 69 and 70 dosed orally at 20, 50

and 100 mg/kg were also listed in Table 7 (n ¼ 3). These data dis-

played a dose-dependent trend. For example, compound 69

relatively moderate bioavailability (F ¼ 31.4%) in rats after oral

administration of 50 mg/kg. Compound 70 had similar but slightly

higher T_max(38.66 4.3 min), C_max(2.04 0.24 mg/mL), AUC

(156.6 21.32 mg∙min/L) and bioavailability (F ¼ 34.8%) at the

same oral dose.

3.4. Tissue distribution

On the basis of this promising proﬁle, the distribution of 70 in

various tissues such as heart, liver, brain, spleen, lungs and kidney

was analyzed with 50 mg/kg oral dose in rats. As shown in Fig. 8,

the C_maxreached the highest value in organs such as brain, heart,

Table 7

Pharmacokinetic parameters of selected compound 69 and 70 in rat (n ¼ 3).

Dose (mg/kg)

Compound 69

Compound 70

20 (Iv.)

20 (Po.)

50 (Po.)

100 (Po.)

20 (Iv.)

20 (Po.)

50 (Po.)

100 (Po.)

T_1/2(min)

19.72 4.2

N/A

55.83 5.6

35.25 4.5

0.39 0.06

8.94 0.47

0.88 0.33

72.80 4.81

41.3

58.48 6.4

35.56 5.5

0.48 0.06

9.26 0.67

1.89 0.58

138.4 10.4

31.4

56.96 7.2

36.16 5.5

0.56 0.08

9.98 0.49

3.77 0.86

306.4 46.2

34.8

22.80 3.2

N/A

57.88 8.4

37.14 6.5

0.38 0.04

9.86 0.68

0.98 0.42

78.22 5.14

43.4

59.24 7.8

38.66 4.3

0.45 0.06

10.17 0.84

2.04 0.24

156.6 21.32

34.8

58.48 8.9

38.78 5.9

0.52 0.16

11.26 0.87

3.98 0.22

328.6 54.48

36.5

T_max(min)

CL (L/min/kg)

V_ss(L/kg)

C_max(mg/mL)

AUC (mg∙min/L)

0.16 0.01

1.98 0.28

6.22 0.76

176.3 16.2

N/A

0.14 0.006

2.14 0.28

6.48 0.46

180.2 18.2

N/A

Oral bioavailability (% F)

14

Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

Fig. 8. Tissue distribution of compound 70 in SD rat heart, liver, brain, spleen, lungs, and kidney after a 50 mg/kg oral dose (n ¼ 3).

and kidney at 60 min after oral administration of 70 and were about

2 times higher in the spleen and lungs. That means compound 70

could easily permeate through blood brain barrier to the brain and

stayed there for a period of time.

crystal structure of LDH and its complexes with small molecular

inhibitors were available from Protein Databank (PDB code: 4JNK

[63]).

As shown in Figs. 10 and 11, 70 and ligand compound 22 ((2R)-2-

{[5-cyano-4-(3,4-dichlorophenyl)-6-oxo-1,6-dihydropyrimidin-2-

yl]sulfanyl}2-N-(4-sulfamoylphenyl) propanamide) [63] of LDH

could nearly overlap in the binding model. The compound 70

bound to the protein near several conserved residues involved in

the catalytic processing of LDH substrates (Argꢀ105, Tyrꢀ238 and

Thrꢀ247), which the Argꢀ105 was the key mobile residue in the

enzyme's catalytic cycle [64]. The presence of 70 resulted in a

relatively ‘open’ conformation of this region in which a fourth

conserved residue required for enzyme catalysis (Argꢀ105) was

positioned well-removed from the substrate binding site. Analysis

of 70's binding mode in the active binding site demonstrated that

the docking mode of the 70 was similar to the ligand compound

with the same H-bond between Argꢀ105 and Thrꢀ247. The

3.5. Metabolite identiﬁcation

Metabolite identiﬁcation work was undertaken to establish the

metabolic activity exerted upon compound 70 (Fig. 9 and Table 8).

In the study, six metabolites were detected in the urine and plasma

of SD rats dosed with 70. These metabolites were named as M1

through to M6 based on their eluting time under HPLC conditions.

Among the six metabolites, M6 was identiﬁed as compound 22, M3

was identiﬁed as compound 3, M5 was identiﬁed as compound 18,

M4 was identiﬁed as cis-isomer of compound 18, and M2 and M1

were identiﬁed as glucuronic acid (GluA) ester of compound 22 and

compound 18 respectively. Location of the GluA groups was

established through mass spectrometry fragmentation patterns.

M1 to M6 were detected both in urine and plasma samples.

methoxyl group of compound 70 in the a-asaronol moiety formed a

hydrogen bond with NH₂group of the LDH Argꢀ105 residue

(ꢀO/HN, 1.99 Å). And the methoxyl group of cinnamic acid moiety

formed another hydrogen bond with NH group of LDH Thrꢀ247

residue (ꢀO/HN, 2.68 Å). In addition, the carbonyl present in 70

was located within H-bond distance of the LDH Tyrꢀ238 backbone

amide OH group (ꢀO/HO, 2.53 Å). These H-bond interactions at

the positions might favor the binding activity and played an

important role in the inhibitory potency of 70 against LDH ac-

cording to the docking results. The Surﬂex dock scores of 70 and

ligand compound were 11.564 and 9.846 respectively, which could

3.6. Molecular modeling

Based on the inhibition results, we selected compound 70, one

of our best LDH inhibitors in our study, as a ligand example. To

explore the binding modes of 70 with the active sites of LDH, mo-

lecular docking was performed using Surﬂex-Dock (SYBYL-X 2.0,

Tripos Inc. St. Louis, USA) which was used to explore the probable

binding conformation and results shown in docking scores. The

Fig. 9. Metabolic pathways of compound 70 in SD rats urine and plasma.

Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

15

Table 8

Identiﬁed metabolites of compound 70 in SD rats urine and plasma (MS).

Peak ID

Mass shift

Found m/z

Biotransformation

RT (min)

Relative MS abundance

Urine

plasma

70

þ1

475

437

467

247

239

269

parent

40.6

50.1

55.2

67.5

78.7

79.8

81.4

0.3 ꢂ 10⁵

4.9 ꢂ 10⁵

1.7 ꢂ 10⁶

0.6 ꢂ 10⁵

2.2 ꢂ 10⁶

0.9 ꢂ 10⁷

0.7 ꢂ 10⁵

0.2 ꢂ 10⁵

0.1 ꢂ 10⁵

0.5 ꢂ 10⁵

1.1 ꢂ 10⁶

2.9 ꢂ 10⁷

0.2 ꢂ 10⁵

M1

M2

M3

M4

M5

M6

þ23

þ1

glucuronidation

hydrolysis

isomerization

oxidation

þ1

hydrolysis

STP. Among them, representative compound 70 exhibited good

modulatory activity at human 2 GABA_Areceptors (EC₅₀

46.3 7.3 M). Thus, some of these compounds could be potentially

a1b2g

m

developed as novel AEDs, especially for some of refractory epi-

lepsies such as Dravet syndrome. In the meanwhile, further

mechanistic studies concerning function of substituted cinnamic a-

asaronol esters are underway in which 68e70 and 75 serve as

important test substrates.

More importantly, the above studies once again exempliﬁed

the effectiveness and rationality of CTCMMC strategy, which not

only provided a more advanced point of entry for further studies

on TCM, but also allowed rapid access to design simpler struc-

tures with comparable or superior activities to that of their parent

TCM.

Fig. 10. Docking conﬁrmation of compound 70 (green) and ligand compound

(magenta, compound 22 in ref. [63]) with LDH. (For interpretation of the references to

color in this ﬁgure legend, the reader is referred to the Web version of this article.)

5. Experimental section

5.1. Chemistry

5.1.1. General methods

Reactions were run using commercially available starting

materials without further puriﬁcation. All solvents were analyt-

ical grade, and anhydrous reactions were performed in oven-

dried glassware under an atmosphere of nitrogen. The nuclear

magnetic resonance spectra (¹H NMR and ¹³C NMR) were recor-

ded on Varian Gemini 2000 (600 MHz) or Bruker (500 MHz) in

DMSO‑d₆or CDCl₃using TMS as an internal standard. The

chemical shifts

d were expressed in parts per million (ppm). The

following abbreviations were used for interpreting the spectra: s,

singlet; d, doublet; t, triplet; q, quadruplet; quint, quintuplet;

sext, sextuplet; m, multiplet; dd, doublet of doublets; br, broad

singlet. Mass spectra were obtained with Agilent 1200 series

HPLC-6520 Q-TOF. Reactions were monitored by analytical thin-

layer chromatography (TLC) plates (Merck 0.2 mm precoated sil-

ica gel aluminum sheets (60 F-254)) and analyzed with 254 nm

UV light. The mixtures were puriﬁed by ﬂash column chroma-

tography using silica gel (Qingdao Haiyang Chemical Co., Ltd.

200e300 mesh). Evaporation of solvents was carried out under

reduced pressure at a temperature below 50 ^ꢁC. After column

chromatography, appropriate fractions were pooled, evaporated,

and dried at high vacuum for at least 4 h to give the desired

products in high purity. The purity of the target compounds was

determined by HPLC analysis, using an Agilent 1100 liquid chro-

matograph (UV detection at 254 or 280 nm) and a C₁₈column

Fig. 11. Docking conﬁrmation of compound 70 with LDH-A (Resolution: 1.9 Å, PDB

code: 4JNK). Selected protein residues are shown. Hydrogen bonding interactions with

the protein are indicated as dashed lines.

be inferred that the rational of the program, and the docking results

were reliable.

4. Conclusions

In light of Polygala tenuifolia-Acori Tatarinowii herbal pair and

CTCMMC strategy, we have synthesized 33 novel substituted cin-

namic a-asaronol esters and analogues in this study. The structures

of representative compounds 65 and 78 have been established by

X-ray crystallography. Additionally, animal tests revealed that

compounds 69, 70, and 75 possessed a better anticonvulsant ac-

tivity with less neurotoxicity in comparison to that of previous lead

68, parent compounds (3 and 20) and some currently known AEDs

such as STP. Compound 69e71, and 75 signiﬁcantly attenuated

neuropathic pain in the mice formalin model. Especially, compound

68e70 showed better inhibitory activity against LDH than that of

(250 mm ꢂ 4.6 mm,

5 mm, Zorbax). The mobile phase was a

MeOH/H₂O (80/20) in 25 min; the ﬂow rate was 0.6 mL/min.

Infrared spectra were recorded with a Thermo infrared spec-

trometer (Nicolet 5700). Melting points were determined in open

capillary tubes using a Shanghai Yice melting point apparatus

(WRS-1B) and were uncorrected. All compounds tested for bio-

logical activity showed ꢃ95% purity by HPLC analysis (detection

at 254 nm) and NMR data.

16

Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

5.1.2. (E)-3-(2,4,5-Trimethoxyphenyl)prop-2-en-1-ol (3)

3.83 (s, 3H), 3.83 (s, 4H). HRMS (þESI) 226.1427 [M þ H]^þ(calcd. for

To a stirred solution of methyl (E)-3-(2,4,5-trimethoxyphenyl)

acrylate (38) (100 g, 0.396 mol) in anhydrous THF (800 mL)

at ꢀ60 ^ꢁC was added, over a 30 min period a solution of DIBAL-H

(500 mL of a 1.5 M solution in hexane, 7.25 mol) in anhydrous

THF (1000 mL). The reaction mixture was warmed at ꢀ45 ^ꢁC and

stirred for an additional 2 h. Then the reaction was quenched with

saturated NH₄Cl solution and the mixture was stirred at room

temperature for 30 min. The resulting precipitated inorganic salts

were removed by ﬁltration and washed with EtOAc. The aqueous

layer was extracted with EtOAc (3 ꢂ 200 mL), and the combined

organic layers were dried over anhydrous Na₂SO₄and concentrated

in vacuo. The crude residue was puriﬁed by ﬂash chromatography

on silica gel (petroleum ether/ethyl acetate, 100:1e5:1) to afford

80.8 g of 3 as a light yellow solid. Yield: 91.0%. Mp 65.1e65.6 ^ꢁC. TLC:

R_f¼ 0.42 (1:1 EtOAc/hexanes). ¹H NMR (600 MHz, Chloroform-d)

C

₁₂H₂₀NO^þ₃226.1438).

5.1.6. (E)-2,6-Dimethoxycinnamic acid (13)

Step 1. The intermediate of methyl (E)-3-(2,6-dimethoxyphenyl)

acrylate was prepared using the same conditions as those used for

the preparation of 38 except that 2,6-dimethoxybenzaldehyde 40

(3.3 g,

20.0 mmol)

was

used

in

place

of

2,4,5-

trimethoxybenzaldehyde 37. White solid. Yield: 3.95 g, 89.0%. Mp

75.5e75.8 ^ꢁC (lit [65]. mp 89e90 ^ꢁC). TLC: R_f¼ 0.75 (1:2 EtOAc/

hexanes). ¹H NMR (600 MHz, Chloroform-d)

d

8.14 (d, J ¼ 16.3 Hz,

1H), 7.25 (t, J ¼ 8.4 Hz, 1H), 6.89 (d, J ¼ 16.4 Hz, 1H), 6.55 (d,

J ¼ 8.4 Hz, 2H), 3.87 (s, 6H), 3.79 (s, 3H). ¹³C NMR (151 MHz, Chlo-

roform-d)

d 169.09, 160.09, 135.71, 131.32, 120.31, 112.25, 103.73,

55.82, 51.51. HRMS (þESI) 223.0965 [M þ H]^þ, 245.0788 [M þ Na]^þ

(calcd. for C₁₂H₁₅O₄^þ223.0965 and C₁₂H₁₄O₄Na^þ245.0784).

d

6.97 (s, 1H), 6.86 (d, J ¼ 16.0 Hz, 1H), 6.48 (s, 1H), 6.24 (dt, J ¼ 6.1,

15.9 Hz,1H), 4.30 (d, J ¼ 6.0 Hz, 2H), 3.88 (s, 3H), 3.85 (s, 3H), 3.81 (s,

3H). ¹³C NMR (151 MHz, Chloroform-d)

151.45, 149.60, 143.29,

Step 2. Following a procedure similar to that described for the

preparation of compound 10 (step 2) except that methyl (E)-3-(2,6-

dimethoxyphenyl)acrylate (2.4 g, 10.8 mmol) was used in place of

2,3,4-trimethoxyphenyl formate, the title compound 13 was ob-

tained as a white solid (2.2 g). Yield: 97.8%. Mp 135.8e136.0 ^ꢁC (lit

[66]. mp 79.5e81.4 ^ꢁC). TLC: R_f¼ 0.15 (1:4 EtOAc/hexanes). ¹H NMR

d

126.96, 125.96, 117.34, 109.86, 97.49, 56.66, 56.51, 56.13. IR (KBr)

3345, 3257, 3000, 2935, 2857, 2826, 1613, 1520, 1465, 1454, 1404,

1335, 1318, 1286, 1268, 1210, 1125, 1094, 1030, 968, 869, 859, 813.

HRMS (þESI) 247.0941 [M

þ

Na]^þ(calcd. for C₁₂H₁₆O₄Na^þ

(600 MHz, DMSO‑d₆)

d

7.91 (d, J ¼ 16.3 Hz, 1H), 7.34 (t, J ¼ 8.4 Hz,

247.0944).

1H), 6.75e6.69 (m, 3H), 3.85 (s, 6H). ¹³C NMR (151 MHz, DMSO‑d₆)

d

168.93, 159.47, 134.25, 131.68, 121.48, 111.05, 104.15, 55.93. HRMS

5.1.3. 3-(2,4,5-Trimethoxyphenyl)propan-1-ol (4)

(-ESI) 207.0661 [M - H]^þ(calcd. for C₁₁H₁₁O^þ₄207.0663).

To an EtOH solution (15 mL) of compound 3 (1.12 g, 5.0 mmol) in

a glass bottle was added Pd-C (Pd content 5%, wet, 0.20 g). The

mixture was stirred at room temperature under H₂(1 atm) for 20 h.

The catalyst was ﬁltered with the aid of a Celite pad, and the ﬁltrate

was concentrated under vacuum to obtain 4 (1.08 g) as a white

solid, which could be used without further puriﬁcation. Yield: 96%.

Mp 44.5e44.8 ^ꢁC. TLC: R_f¼ 0.42 (1:1 EtOAc/hexanes). ¹H NMR

5.1.7. (E)-2,3,6-Trimethoxycinnamic acid (17)

Step 1. The intermediate of methyl (E)-3-(2,3,6-

trimethoxyphenyl)acrylate was prepared using the same condi-

tions as those used for the preparation of 38 except that 2,3,6-

trimethoxybenzaldehyde 42 (3.9 g, 20.0 mmol) was used in place

of 2,4,5-trimethoxybenzaldehyde 37. Light yellow oil. Yield: 4.0 g,

80.0%. Mp 50.9e51.1 ^ꢁC. TLC: R_f¼ 0.62 (1:5 EtOAc/hexanes). ¹H

(600 MHz, Chloroform-d)

3.81 (s, 3H), 3.80 (s, 3H),3.56 (t, J ¼ 3.8 Hz, 2H), 2.65 (t, J ¼ 4.8 Hz,

2H), 1.86e1.74 (m, 2H). ¹³C NMR (151 MHz, Chloroform-d)

151.40,

d 6.68 (s, 1H), 6.51 (s, 1H), 3.86 (s, 3H),

NMR (600 MHz, Chloroform-d)

d

8.01 (d, J ¼ 16.3 Hz, 1H), 6.94 (d,

d

J ¼ 16.4 Hz, 1H), 6.88 (d, J ¼ 9.0 Hz, 1H), 6.60 (d, J ¼ 9.0 Hz, 1H), 3.84

143.13, 121.30, 114.07, 97.67, 56.63, 56.60, 56.27, 33.28, 25.37. HRMS

(þESI) 227.1275 [M þ H]^þ, 249.1097 [M þ Na]^þ(calcd. for C₁₂H₁₉O₄^þ

227.1278 and C₁₂H₁₈O₄Na^þ249.1097).

(s, 3H), 3.83 (s, 6H), 3.80 (s, 3H). ¹³C NMR (151 MHz, Chloroform-d)

d

168.81, 153.75, 149.88, 147.13, 135.94, 121.88, 118.14, 114.57, 105.89,

61.01, 56.63, 56.01, 51.67. HRMS (þESI) 253.1070 [M þ H]^þ,

275.0895 [M

C

þ

Na]^þ(calcd. for C₁₃H₁₇O^þ₅253.1071 and

5.1.4. (E)-3-(2,4,5-Trimethoxyphenyl)prop-2-en-1-amine (5)

₁₃H₁₆O₅Na^þ275.0890).

Step 2. Following a procedure similar to that described for the

To an EtOH solution (70 mL) of compound 39 (2.0 g, 5.6 mmol)

in a glass bottle was added hydrazine hydrate (con. 80%, 1 g,

15.8 mmol). After the reaction mixture was stirred at reﬂux tem-

perature for 2 h, the EtOH was evaporated. The residue was

dispersed in the EtOAc (50 mL) and undissolved substance was

ﬁltered out. The ﬁltrate was added water (50 mL) and 10% HCl so-

lution until pH 3 was reached. The aqueous phase was removed and

organic phase was dried on Na₂SO₄, then concentrated in vacuo to

afford crude compound 5, which was puriﬁed by ﬂash chroma-

tography (SiO₂; 100:0ꢀ10:1 DCM/MeOH, v/v) to give a faint yellow

clear oil 1.07 g. Yield: 84.9%. TLC: R_f¼ 0.46 (10:1 DCM/MeOH).

HRMS (þESI) 207.1030 [M e NH₃]^þ, (calcd. for C₁₂H₁₄O^þ₃207.1016).

preparation of compound 45 (step 2) except that methyl (E)-3-

(2,3,6-trimethoxyphenyl)acrylate (3.0 g, 11.9 mmol) was used in

place of 2,3,4-trimethoxyphenyl formate, the title compound 17

was obtained as

a white solid (2.4 g). Yield: 85.7%. Mp

182.4e182.7 ^ꢁC. TLC: R_f¼ 0.10 (1:5 EtOAc/hexanes). ¹H NMR

(600 MHz, DMSO‑d₆)

d

7.80 (d, J ¼ 16.3 Hz, 1H), 7.09 (d, J ¼ 9.1 Hz,

1H), 6.77 (t, J ¼ 13.0 Hz, 2H), 3.81 (s, 3H), 3.77 (s, 3H), 3.74 (s, 3H).

¹³C NMR (151 MHz, DMSO‑d₆)

d

168.44, 152.92, 148.74, 146.53,

134.81, 122.36, 116.66, 115.21, 106.45, 60.37, 56.26, 55.98. HRMS

(-ESI) 237.0765 [M - H]^þ(calcd. for C₁₂H₁₃O^þ₅237.0768).

5.1.8. (E)-2,4,6-Trimethoxycinnamic acid (19)

5.1.5. 3-(2,4,5-Trimethoxyphenyl)propan-1-amine (6)

Step 1. The intermediate of methyl (E)-3-(2,4,6-

trimethoxyphenyl)acrylate was prepared using the same condi-

tions as those used for the preparation of 38 except that 2,4,6-

trimethoxybenzaldehyde 43 (3.9 g, 20.0 mmol) was used in place

of 2,4,5-trimethoxybenzaldehyde 37. White solid. Yield: 3.3 g,

66.0%. Mp 136.1e136.5 ^ꢁC (lit [65]. mp 138e139 ^ꢁC). TLC: R_f¼ 0.70

To an EtOH solution (15 mL) of compound 5 (1.12 g, 5.0 mmol) in

a glass bottle was added Pd-C (Pd content 5%, wet, 0.20 g). The

mixture was stirred at room temperature under H₂(1 atm) for 14 h.

The catalyst was ﬁltered with the aid of a Celite pad, and the ﬁltrate

was concentrated under vacuum to obtain 6 (1.06 g) as a colorless

oil, which can be used in the next step without further puriﬁcation.

Yield: 96%. TLC: R_f¼ 0.52 (5:1 DCM/MeOH). ¹H NMR (500 MHz,

(1:2 EtOAc/hexanes). ¹H NMR (600 MHz, Chloroform-d)

d 8.08 (d,

J ¼ 16.2 Hz, 1H), 6.75 (d, J ¼ 16.3 Hz, 1H), 6.10 (s, 2H), 3.86 (s, 6H),

Chloroform-d)

d

6.86 (s, 1H), 6.53 (s, 1H), 6.08 (ddd, J ¼ 5.6, 10.3,

3.83 (s, 3H), 3.77 (s, 3H). ¹³C NMR (151 MHz, Chloroform-d)

17.2 Hz, 1H), 5.24 (dt, J ¼ 1.6, 17.2 Hz, 1H), 5.11 (dt, J ¼ 1.5, 10.3 Hz,

d 169.54, 162.89, 161.38, 135.79, 117.23, 105.91, 90.52, 55.81, 55.48,

1H), 4.80 (d, J ¼ 5.5 Hz, 1H), 3.89 (s, 1H), 3.88 (s, 3H), 3.86 (s, 1H),

51.43. HRMS (þESI) 253.1071 [M þ H]^þ, 275.0893 [M þ Na]^þ(calcd.

Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

17

for C₁₃H₁₇O^þ₅253.1071 and C₁₃H₁₆O₅Na^þ275.0890).

5.1.11. (E)-2,3,5,6-Tetramethoxycinnamic acid (23)

Step 2. Following a procedure similar to that described for the

preparation of compound 45 (step 2) except that methyl (E)-3-

(2,4,6-trimethoxyphenyl)acrylate (2.3 g, 9.1 mmol) was used in

place of 2,3,4-trimethoxyphenyl formate, the title compound 19

Step 1. The intermediate of methyl (E)-3-(2,3,5,6-

tetramethoxyphenyl)acrylate was prepared using the same condi-

tions as those used for the preparation of 38 except that 2,3,5,6-

tetramethoxybenzaldehyde 54 (4.52 g, 20.0 mmol) was used in

place of 2,4,5-trimethoxybenzaldehyde 37. White solid. Yield:

3.96 g, 70.1%. Mp 77.3e77.5 ^ꢁC. TLC: R_f¼ 0.72 (1:2 EtOAc/hexanes).

Step 2. Following a procedure similar to that described for the

preparation of compound 45 (step 2) except that methyl (E)-3-

(2,3,5,6-tetramethoxyphenyl)acrylate (2.82 g, 10.0 mmol) was used

in place of 2,3,4-trimethoxyphenyl formate, the title compound 23

was obtained as a light kelly solid (2.45 g). Yield: 91.3%. Mp

182.2e182.4 ^ꢁC. TLC: R_f¼ 0.65 (100% EtOAc). ¹H NMR (600 MHz,

was obtained as

a white solid (1.8 g). Yield: 81.8%. Mp

232.8e232.9 ^ꢁC. TLC: R_f¼ 0.12 (1:2 EtOAc/hexanes). ¹H NMR

(600 MHz, DMSO‑d₆)

d

7.85 (d, J ¼ 16.2 Hz, 1H), 6.55 (d, J ¼ 16.2 Hz,

1H), 6.28 (s, 2H), 3.85 (s, 6H), 3.83 (s, 3H). ¹³C NMR (151 MHz,

DMSO‑d₆)

d 169.18, 162.68, 160.75, 134.59, 117.79, 104.45, 90.95,

55.95, 55.52. HRMS (-ESI) 237.0765 [M - H]^þ(calcd. for C₁₂H₁₃O^þ₅

237.0768).

DMSO‑d₆)

d

7.71 (d, J ¼ 16.3 Hz, 1H), 6.88 (s, 1H), 6.77 (d, J ¼ 16.3 Hz,

5.1.9. (E)-2,3,4,5-Tetramethoxycinnamic acid (21)

1H), 3.83 (s, 6H), 3.66 (s, 6H). ¹³C NMR (151 MHz, DMSO‑d₆)

Step 1. The intermediate of methyl (E)-3-(2,3,4,5-

tetramethoxyphenyl)acrylate was prepared using the same condi-

tions as those used for the preparation of 38 except that 2,3,4,5-

tetramethoxybenzaldehyde 43 (4.5 g, 20.0 mmol) was used in

place of 2,4,5-trimethoxybenzaldehyde 37. White solid. Yield: 4.4 g,

77.2%. Mp 62.3e62.6 ^ꢁC. TLC: R_f¼ 0.68 (1:2 EtOAc/hexanes). ¹H

d

168.14, 148.79, 141.17, 135.03, 123.10, 121.17, 101.70, 60.23, 56.35.

HRMS (-ESI) 267.0878 [M - H]^þ(calcd. for C₁₃H₁₅O^þ₆267.0874).

5.1.12. (E)-2,3,4,5,6-Pentamethoxycinnamic acid (24)

Step 1. The intermediate of methyl (E)-3-(2,3,4,5,6-

pentamethoxyphenyl)acrylate was prepared using the same con-

ditions as those used for the preparation of 38 except that 2,3,4,5,6-

pentamethoxybenzaldehyde 48 (5.13 g, 20.0 mmol) was used in

place of 2,4,5-trimethoxybenzaldehyde 37. Colorless oil. Yield:

4.53 g, 72.6%. TLC: R_f¼ 0.55 (1:2 EtOAc/hexanes). ¹H NMR

NMR (600 MHz, Chloroform-d)

d

7.91 (d, J ¼ 16.1 Hz, 1H), 6.78 (s,

1H), 6.40 (d, J ¼ 16.1 Hz, 1H), 3.92 (s, 3H), 3.92 (s, 3H), 3.85 (s, 3H),

3.84 (s, 3H), 3.80 (s, 3H). ¹³C NMR (151 MHz, Chloroform-d)

d

167.75,

149.79, 147.73, 147.34, 145.41, 139.50, 122.73, 117.81, 104.53, 61.94,

61.40, 61.33, 56.30, 51.79. HRMS (þESI) 283.1176 [M þ H]^þ,

305.0998 [M þ Na]^þ(calcd. for C₁₄H₁₉O₆^þ283.1176 and C₁₄H₁₈O₆Na^þ

305.0996).

Step 2. Following a procedure similar to that described for the

preparation of compound 45 (step 2) except that methyl (E)-3-

(2,3,4,5-tetramethoxyphenyl)acrylate (2.8 g, 10.0 mmol) was used

in place of 2,3,4-trimethoxyphenyl formate, the title compound 21

(600 MHz, Chloroform-d)

d

7.84 (d, J ¼ 16.3 Hz, 1H), 6.85 (d,

J ¼ 16.3 Hz, 1H), 3.95 (s, 3H), 3.84 (s, 6H), 3.81 (s, 6H), 3.77 (s, 3H).

¹³C NMR (151 MHz, Chloroform-d)

d 149.51, 149.34, 143.21, 135.70,

120.72, 61.53, 61.31, 61.12, 51.63.HRMS (þESI) 313.1282 [M þ H]^þ,

335.1107 [M þ Na]^þ(calcd. for C₁₅H₂₁O₇^þ313.1282 and C₁₅H₂₀O₇Na^þ

335.1101).

was obtained as

a

white solid (2.5 g). Yield: 89.0%. Mp

Step 2. Following a procedure similar to that described for the

preparation of compound 45 (step 2) except that methyl (E)-3-

(2,3,4,5,6-pentamethoxyphenyl)acrylate (3.12 g, 10.0 mmol) was

used in place of 2,3,4-trimethoxyphenyl formate, the title com-

pound 24 was obtained as a white solid (2.87 g). Yield: 96.2%. Mp

141.1e141.2 ^ꢁC (lit [67]. mp 138e139 ^ꢁC) TLC: R_f¼ 0.62 (100%

108.7e108.9 ^ꢁC. TLC: R_f¼ 0.88 (100% EtOAc). ¹H NMR (600 MHz,

DMSO‑d₆)

d

12.36 (s, 1H), 7.72 (d, J ¼ 16.1 Hz, 1H), 7.12 (s, 1H), 6.56

(d, J ¼ 16.1 Hz,1H), 3.82 (s, 3H), 3.82 (s, 3H), 3.79 (s, 3H), 3.75 (s, 3H).

¹³C NMR (151 MHz, DMSO‑d₆)

d

167.79, 149.60, 146.63, 146.58,

144.66, 137.78, 122.17, 119.19, 104.89, 61.62, 60.98, 60.71, 56.16.

HRMS (-ESI) 267.0882 [M - H]^þ(calcd. for C₁₃H₁₅O^þ₆267.0874).

EtOAc). ¹H NMR (600 MHz, DMSO‑d₆)

d

7.67 (d, J ¼ 16.3 Hz,1H), 6.69

(d, J ¼ 16.3 Hz, 1H), 3.90 (s, 3H), 3.79 (s, 6H), 3.76 (s, 6H). ¹³C NMR

(151 MHz, DMSO‑d₆) d 168.29, 149.01, 148.73, 142.90, 134.53, 121.59,

5.1.10. (E)-2,3,4,6-Tetramethoxycinnamic acid (22)

116.38, 61.15, 61.00, 60.85. HRMS (-ESI) 297.0980 [M - H]^þ(calcd. for

C₁₄H₁₇O^þ₇297.0980).

Step 1. The intermediate of methyl (E)-3-(2,3,4,6-

tetramethoxyphenyl)acrylate was prepared using the same condi-

tions as those used for the preparation of 38 except that 2,3,4,6-

tetramethoxybenzaldehyde 51 (4.5 g, 20.0 mmol) was used in

place of 2,4,5-trimethoxybenzaldehyde 37. White solid. Yield: 4.0 g,

71.2%. Mp 114.4e114.7 ^ꢁC. TLC: R_f¼ 0.58 (1:2 EtOAc/hexanes). ¹H

5.1.13. Methyl (E)-3-(2,4,5-trimethoxyphenyl)acrylate (38)

The Meldrum's acid (54.0 g, 0.37 mol) was dissolved in toluene

(500 mL), and then methanol (20.0 mL, 0.49 mol) was added and

the mixture was stirred at reﬂux temperature for 5 h. The reaction

was cooled to room temperature, added with 2,4,5-

trimethoxybenzaldehyde (37.3 g, 0.19 mol), pyridine (40.0 mL) and

piperidine (4.0 mL). The mixture was stirred at room temperature

for another 24 h and monitored by TLC. The volatile materials were

removed in vacuo. Water was added, and the mixture was extracted

with EtOAc (3 ꢂ 200 mL). The combined organic solution was dried

(MgSO₄) and concentrated to give light yellow crude solid, which

was recrystallized from a mixture of EtOAc/petroleum ether (1:9) to

afford pure product 44.3 g. The procedure was adapted from Xia

et al. [30]. Light yellow solid. Yield: 92.5%. Mp 105.2e105.4 ^ꢁC (lit

[65]. mp 97e98 ^ꢁC). TLC: R_f¼ 0.69 (1:1 EtOAc/hexanes). ¹H NMR

NMR (600 MHz, Chloroform-d)

d

7.97 (d, J ¼ 16.3 Hz, 1H), 6.81 (d,

J ¼ 16.2 Hz, 1H), 6.27 (s, 1H), 3.90 (s, 3H), 3.90 (s, 3H), 3.87 (s, 3H),

3.80 (s, 3H), 3.78 (s, 3H). ¹³C NMR (151 MHz, Chloroform-d)

d

169.22, 156.40, 155.45, 154.48, 136.45, 135.88, 118.71, 110.35, 91.99,

61.28, 61.20, 56.15, 55.98, 51.54. HRMS (þESI) 283.1176 [M þ H]^þ,

305.0998 [M þ Na]^þ(calcd. for C₁₄H₁₉O₆^þ283.1176 and C₁₄H₁₈O₆Na^þ

305.0996).

Step 2. Following a procedure similar to that described for the

preparation of compound 45 (step 2) except that methyl (E)-3-

(2,3,4,6-tetramethoxyphenyl)acrylate (2.8 g, 10.0 mmol) was used

in place of 2,3,4-trimethoxyphenyl formate, the title compound 22

was obtained as

a white solid (2.6 g). Yield: 92.2%. Mp

191.6e192.0 ^ꢁC. TLC: R_f¼ 0.67 (100% EtOAc). ¹H NMR (600 MHz,

(600 MHz, CDCl₃)

6.37 (d, J ¼ 16.1 Hz,1H), 3.92 (s, 3H), 3.87 (s, 3H), 3.86 (s, 3H), 3.79 (s,

3H). ¹³C NMR (150 MHz, CDCl₃)

168.29, 154.01, 152.23, 143.36,

d

7.97 (d, J ¼ 16.1 Hz, 1H), 7.00 (s, 1H), 6.49 (s, 1H),

DMSO‑d₆)

d

12.07 (s, 1H), 7.76 (d, J ¼ 16.2 Hz, 1H), 6.62 (d,

J ¼ 16.2 Hz, 1H), 6.54 (s, 2H), 3.88 (s, 9H), 3.80 (s, 4H), 3.69 (s, 4H).

d

¹³C NMR (151 MHz, DMSO‑d₆)

d

168.82, 155.88, 155.38, 153.37,

139.84, 115.51, 115.03, 111.00, 96.98, 56.57, 56.49, 56.20, 51.65.

135.64, 134.90, 118.93, 108.68, 93.03, 60.96, 60.57, 56.11, 56.05.

HRMS (-ESI) 267.0882 [M - H]^þ(calcd. for C₁₃H₁₅O^þ₆267.0874).

HRMS (þESI) 253.1086 [M þ H]^þ, 275.0895 [M þ Na]^þ(calcd. for

C

₁₃H₁₇O^þ₅253.1071 and C₁₃H₁₆O₅Na^þ275.0890).

18

Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

5.1.14. (E)-2-(3-(2,4,5-Trimethoxyphenyl)allyl)isoindoline-1,3-

dione (39)

trimethoxyphenyl formate (73 g) as colorless oil, which was used

in the next step without further puriﬁcation. TLC: R_f¼ 0.60 (1:5

EtOAc/hexanes).

To a 100 mL glass bottle compound 3 (1.0 g, 4.44 mmol) was

added and dissolved in THF. Phthalimide (0.98 g, 6.66 mmol) and

triphenylphosphine (1.51 g, 5.77 mmol) were then added and the

reaction was cooled to 0 ^ꢁC. Diisopropyl azodicarboxylate (1.17 g,

5.77 mmol) was then added dropwise over 2 min. The reaction was

allowed to warm to room temperature and stirred overnight under

nitrogen. When the reaction was complete by TLC the reaction was

added water (50 mL) and EtOAc (50 mL). The organic phase was

separated and dried on Na₂SO₄, then concentrated in vacuo to

afford semisolid, which was puriﬁed by ﬂash chromatography

(SiO₂; 5:1ꢀ3:1 petroleum ether/ethyl acetate, v/v) to give a white

solid 1.40 g [68]. Yield: 89.2%. Mp 149.6e150.0 ^ꢁC. TLC: R_f¼ 0.68

Step 2. Crude 2,3,4-trimethoxyphenyl formate (73 g) was added

to methanol (120 mL) and treated with aq. 10% NaOH (300 mL) at

0 ^ꢁC. The resulting homogeneous solution was stirred and allowed

to warm to 40 ^ꢁC for 2 h and determined to be complete by TLC.

Methanol was distilled out completely from the reaction mixture

under reduced pressure. The residue was added to H₂O (200 mL),

acidiﬁed with 3 N HCI and extracted with DCM (3 ꢂ 150 mL). The

organic layer was washed with H₂O (2 ꢂ 200 mL), dried over

Na₂SO₄, ﬁltered and concentrated completely under reduced

pressure to yield 2,3,4-trimethoxyphenol (71 g) as a white solid

(wet). TLC: R_f¼ 0.50 (1:5 EtOAc/hexanes). This material was used

for the subsequent reaction without further puriﬁcation.

(1:2 EtOAc/hexanes). ¹H NMR (600 MHz, Chloroform-d)

d 7.84 (dd,

J ¼ 3.0, 5.4 Hz, 2H), 7.70 (dd, J ¼ 3.0, 5.4 Hz, 2H), 6.95 (d, J ¼ 15.9 Hz,

1H), 6.90 (s, 1H), 6.45 (s, 1H), 6.13 (dt, J ¼ 6.7, 15.9 Hz, 1H), 4.43 (d,

J ¼ 6.7 Hz, 2H), 3.87 (s, 3H), 3.82 (s, 3H), 3.80 (s, 3H). ¹³C NMR

Step 3. To a solution of 2,3,4-trimethoxyphenol (71 g, 0.38 mol),

K₂CO₃(50 g, 0.36 mol) in acetone (400 mL) at room temperature

was added dimethvl sulfate (53 g, 0.42 mol) in 15 min. The reaction

mixture was stirred and reﬂuxed for 20 h, and the solvent was

evaporated followed by the addition of water (400 mL). The

mixture was simply stirred for 15 min and allowed to stand at room

temperature for 4 h. The crude crystalline product formed and was

collected, washed with water and puriﬁed by recrystallization from

MeOH to afford 1,2,3,4-tetramethoxybenzene 45 (42 g) as white

solid. Total yield (from step 1 to 3): 70.6%. Mp 85.8e86.1 ^ꢁC (lit [70].

mp 87e87.5 ^ꢁC). TLC: R_f¼ 0.75 (1:5 EtOAc/hexanes). ¹H NMR

(151 MHz, Chloroform-d)

d 168.17, 151.56, 149.66, 143.19, 134.02,

132.29, 128.54, 123.34, 120.92, 116.88, 109.68, 97.41, 56.67, 56.43,

56.08, 40.40. HRMS (þESI) 354.1319 [M þ H]^þ, 376.1135 [M þ Na]^þ

(calcd. for C₂₀H₂₀NO₅^þ354.1336 and C₂₀H₁₉NO₅Na^þ376.1155).

5.1.15. 2,3,6-Trimethoxybenzaldehyde (42)

n-Butyl lithium in hexane 45 mL (2.5 M, 0.11 mol) was added

dropwise to a solution of 1,2,4-trimethoxybenzene 41 (16.8 g,

0.10 mol) in anhydrous THF (160 mL) at ꢀ60 ^ꢁC under a nitrogen

atmosphere (about 30 min). A white precipitate was formed and

temperature raised to ꢀ50 ^ꢁC. After completion of adding n-Butyl

lithium, the reaction mixture was stirred additionally for 1.5 h

between ꢀ40 ^ꢁC and -60 ^ꢁC. Then, anhydrous DMF (8.7g, 0.12 mol)

was added dropwise at ꢀ60 ^ꢁC. During this addition, white pre-

cipitate was disappear. The mixture was allowed to stir at ꢀ40 ^ꢁC

for 2 h (monitored by TLC), then cautiously quenched with satu-

rated NH₄Cl solution and followed by stirring at room temperature

for another 30 min. The reaction was extracted with EtOAc

(3 ꢂ 150 mL) and saturated NH₄Cl solution (250 mL). The total

organic layer was washed with brine (3 ꢂ 150 mL), dried over

anhydrous Na₂SO₄, ﬁltered, and concentrated under reduced

pressure to obtain a crude residue, which was subjected to ﬂash

chromatography on silica gel (petroleum ether/dichloromethane/

ethyl acetate, 9:1:1e7:1:1e6:1:1) to afford 8.4 g of 42 as a light

yellow solid. Yield: 42.9%. Mp 28.2e28.5 ^ꢁC (lit [69]. mp 20 ^ꢁC). TLC:

R_f¼ 0.55 (1:1 EtOAc/hexanes). ¹H NMR (600 MHz, Chloroform-d)

(600 MHz, Chloroform-d) d C

6.58 (s, 2H), 3.90 (s, 6H), 3.82 (s, 6H). ¹³

NMR (151 MHz, Chloroform-d) d 147.91, 143.52, 106.53, 61.33, 56.52.

HRMS (þESI) 199.0968 [M þ H]^þ, 221.0788 [M þ Na]^þ(calcd. for

C

₁₀H₁₅O^þ₄199.0965 and C₁₀H₁₄O₄Na^þ221.0784).

5.1.17. 2,3,4,5-Tetramethoxybenzaldehyde (46)

1,2,3,4-tetramethoxybenzene 45 (8.2 g, 41.4 mmol) and hexa-

methylenetetramine (7.0 g, 50.0 mmol) were dissolved in tri-

ﬂuoroacetic acid (110 mL). The mixture was stirred and heated to

70 ^ꢁC for 8 h. After the completion of reaction monitored by TLC, the

reaction mixture was condensed under reduced pressure and

neutralized with cold saturated NaHCO₃solution to pH ~7. The

desired product was extracted with DCM (2 ꢂ 150 mL), washed

with brine (2 ꢂ 150 mL), dried over anhydrous sodium sulfate,

concentrated, and puriﬁed by silica-gel ﬂash chromatography

(petroleum ether/dichloromethane/ethyl acetate, 9:1:1) to afford

2,3,4,5-tetramethoxybenzaldehyde (8.4 g) as light yellow solid.

Yield: 89.4%. Mp 35.2e35.5 ^ꢁC (lit [71]. mp 37e39 ^ꢁC) TLC: R_f¼ 0.62

[41]

d

10.42 (s, 1H), 7.06 (d, J ¼ 9.1 Hz, 1H), 6.62 (d, J ¼ 9.1 Hz, 1H),

(1:5 EtOAc/hexanes). ¹H NMR (600 MHz, Chloroform-d) [72]

d

10.27

(s,1H), 7.09 (s,1H), 3.97 (s, 3H), 3.95 (s, 3H), 3.92 (s, 3H), 3.85 (s, 3H).

¹³C NMR (151 MHz, Chloroform-d)

188.89, 152.42, 149.92, 149.42,

3.89 (s, 3H), 3.82 (s, 3H), 3.82 (s, 3H). ¹³C NMR (151 MHz, Chloro-

form-d) d 189.80,154.93,152.14,146.87,119.74,119.21,106.46, 62.08,

d

56.79, 56.28. HRMS (þESI) 197.0809 [M þ H]^þ, 219.0627 [M þ Na]^þ

146.72, 124.11, 103.82, 62.97, 61.39, 61.37, 56.23. HRMS (þESI)

227.0914 [M þ H]^þ, 249.0734 [M þ Na]^þ(calcd. for C₁₁H₁₅O₅^þ

227.0914 and C₁₁H₁₄O₅Na^þ249.0733).

(calcd. for C₁₀H₁₃O₄^þ197.0808 and C₁₀H₁₂O₄Na^þ219.0628).

5.1.16. 1,2,3,4-Tetramethoxybenzene (45)

Step 1. In a 2 L three-necks ﬂask was added with 2,3,4-

trimethoxybenzaldehyde 44 (58.9 g, 0.3 mol) followed by anhy-

drous DCM (1.2 L) and cooled to 0e5 ^ꢁC. To this solution, m-CPBA

(75%, 89.8 g, 0.39 mol) was then added portion-wise whilst main-

taining temperature of the reaction mixture in the range 0e5 ^ꢁC.

The reaction mixture was allowed to warm to room temperature

and stirred for 24 h to yield a white slurry. The precipitate was

ﬁltered and washed with 200 mL of anhydrous DCM, then the

ﬁltrate was washed with 10% (w/w) sodium bisuﬁte (2 ꢂ 200 mL/

each time for 20 min), 5% aqueous Na₂CO₃(2 ꢂ 200 mL/each time

for 20 min), water (2 ꢂ 200 mL), brine (2 ꢂ 200 mL) until the starch

iodide paper showed no oxidant remained in organic phase. The

organic phase was dried over anhydrous Na₂SO₄, ﬁltered, and

concentrated under reduced pressure to obtain a crude 2,3,4-

5.1.18. 1,2,3,4,5-Pentamethoxybenzene (47)

Following the three steps similar to that described for the

preparation of compound 45 except that an equivalent amount of

2,3,4,5-tetramethoxybenzaldehyde 46 (67.9 g, 0.3 mol) was used in

place of 2,3,4-trimethoxybenzaldehyde 44, the title compound 47

was obtained as a colorless oil, which solidiﬁed to a white solid on

prolonged standing (46.5 g). Total yield: 67.8%. Mp 56.4e56.6 ^ꢁC (lit

[73]. mp 59e61 ^ꢁC). TLC: R_f¼ 0.70 (1:5 EtOAc/hexanes). ¹H NMR

(600 MHz, Chloroform-d)

d

6.29 (s, 1H), 3.94 (s, 3H), 3.84 (s, 6H),

149.17, 147.95,

3.82 (s, 6H). ¹³C NMR (151 MHz, Chloroform-d)

d

136.62, 93.40, 61.51, 56.50. HRMS (þESI) 229.1076 [M þ H]^þ,

251.0888 [M þ Na]^þ(calcd. for C₁₁H₁₇O₅^þ229.1071 and C₁₁H₁₆O₅Na^þ

251.0890).

Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

19

5.1.19. 2,3,4,5,6-Pentamethoxybenzaldehyde (48)

tetramethoxybenzene 53 was used in place of 1,2,3,4-

tetramethoxybenzene 45, the title compound 54 was obtained as

a white solid (8.8 g). Yield: 93.6%. Mp 72.0e72.3 ^ꢁC (lit [66]. mp

79.5e81.4 ^ꢁC). TLC: R_f¼ 0.50 (1:4 EtOAc/hexanes). ¹H NMR

Following a procedure similar to that described for the prepa-

ration of compound 46 except that an equivalent amount of

1,2,3,4,5-pentamethoxybenzene 47 (9.4 g, 41.4 mmol) was used in

place of 1,2,3,4-tetramethoxybenzene 45, the title compound 48

was obtained as a colorless oil (9.5 g). Yield: 89.6%. TLC: R_f¼ 0.60

(600 MHz, Chloroform-d)

d

10.40 (s, 1H), 6.78 (s, 1H), 3.88 (s, 6H),

190.14, 149.38,

3.85 (s, 6H). ¹³C NMR (151 MHz, Chloroform-d)

d

(1:4 EtOAc/hexanes). ¹H NMR (600 MHz, Chloroform-d) [74]

d

10.28

143.86, 143.02, 124.38, 104.85, 100.29, 62.33, 56.85. HRMS (þESI)

227.0913 [M þ H]^þ, 249.0736 [M þ Na]^þ(calcd. for C₁₁H₁₅O₅^þ

227.0914 and C₁₁H₁₄O₅Na^þ249.0733).

(s, 1H), 4.03 (s, 3H), 3.89 (s, 6H), 3.85 (s, 6H). ¹³C NMR (151 MHz,

Chloroform-d)

d 188.69, 153.41, 151.86, 142.94, 118.97, 62.41, 61.58,

61.54. HRMS (þESI) 257.1042 [M þ H]^þ, 279.0837 [M þ Na]^þ(calcd.

for C₁₂H₁₇O^þ₆257.1020 and C₁₂H₁₆O₆Na^þ279.0839).

5.1.24. General method for the preparation of cinnamic acid ester

derivatives (55e85)

5.1.20. 1,2,3,5-Tetramethoxybenzene (50)

A mixture of the compound 3 or 4 (5 mmol), cinnamic acid

derivatives (6 mmol), and DMAP (1 mmol) in DCM (15 mL) was

stirred for 10 min at room temperature. Successively, EDCI

(6e10 mmol) were added quickly in one portion. The reaction

mixture was continually stirred at room temperature for 10e18 h

under nitrogen and monitored by TLC. Then the clear solution

treated with an aqueous saturated NaHCO₃solution containing ice

(60 mL) for 5 min. The aqueous reaction mixture was extracted

with DCM (3 ꢂ 20 mL). The combined extracts were dried with dry

Na₂SO₄and concentrated in vacuo. The crude compound was pu-

riﬁed by ﬂash chromatography (SiO₂; 100:0ꢀ50:50ꢀ0:100 hex-

anes/DCM, v/v). The ﬁnal esters were obtained as solid substances

after concentration of organic solvents under reduced pressure.

Following the three steps similar to that described for the

preparation of compound 45 except that an equivalent amount of

3,4,5-trimethoxybenzaldehyde 49 (58.9 g, 0.3 mol) was used in

place of 2,3,4-trimethoxybenzaldehyde 44, the title compound 50

was obtained as a colorless oil, which solidiﬁed to a white solid on

prolonged standing (40.2 g). Total yield: 67.6%. Mp 69.3e69.4 ^ꢁC (lit

[75]. mp 46 ^ꢁC). TLC: R_f¼ 0.78 (1:4 EtOAc/hexanes). ¹H NMR

(600 MHz, Chloroform-d)

d 6.14 (s, 2H), 3.83 (s, 6H), 3.77 (d,

J ¼ 2.5 Hz, 6H). ¹³C NMR (151 MHz, Chloroform-d)

d 156.33, 153.80,

132.33, 91.71, 61.10, 56.15, 55.61. HRMS (þESI) 199.0970 [M þ H]^þ,

221.0786 [M þ Na]^þ(calcd. for C₁₀H₁₅O₄^þ199.0965 and C₁₀H₁₄O₄Na^þ

221.0784).

5.1.21. 2,3,4,6-Tetramethoxybenzaldehyde (51)

5.1.24.1. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(2-

methoxyphenyl)acrylate (55). Light yellow solid. Yield: 87.5%. Mp

96.4e96.8 ^ꢁC. TLC: R_f¼ 0.56 (1:1 EtOAc/hexanes). HPLC (purity

Phosphorus oxychloride (13.9 g, 90 mmol) was added to anhy-

drous DMF (100 mL) and stirred at a temperature of 0 ^ꢁC for about

15 min under nitrogen atmosphere, followed by addition of 1,2,3,5-

Tetramethoxybenzene 50 (9.0 g, 45 mmol) dropwise over a period

of 30 min. The reaction mixture was stirred for 1 h at 0 ^ꢁC, then

allowed to warm to room temperature and stirred overnight. The

mixture was poured over crushed ice followed by saturated sodium

carbonate solution. The aqueous solution was extracted with

dichloromethane (2 ꢂ 150 mL). The combined organic layers were

washed with brine (2 ꢂ 150 mL). The organic layer was dried over

anhydrous sodium sulfate, and the sodium sulfate was removed by

ﬁltration. The solvent was removed by rotary evaporation to obtain

the light yellow oil which was puriﬁed by silica-gel ﬂash chroma-

97.7%): t_R¼ 7.62 min ¹H NMR (600 MHz, Chloroform-d)

d 8.03 (d,

J ¼ 16.2 Hz,1H), 7.50 (d, J ¼ 7.7 Hz,1H), 7.34 (t, J ¼ 7.8 Hz,1H), 7.00 (s,

1H), 6.99e6.93 (m, 2H), 6.90 (d, J ¼ 8.3 Hz, 1H), 6.57 (d, J ¼ 16.1 Hz,

1H), 6.50 (s, 1H), 6.26 (dt, J ¼ 15.9, 6.7 Hz, 1H), 4.86 (d, J ¼ 6.7 Hz,

2H), 3.89 (s, 3H), 3.87 (s, 3H), 3.86 (s, 3H), 3.83 (s, 3H). ¹³C NMR

(151 MHz, Chloroform-d)

d167.47, 158.46, 151.76, 149.97, 143.40,

140.46, 131.57, 129.08, 129.04, 123.52, 121.76, 120.79, 118.72, 117.11,

111.23, 110.08, 97.62, 65.94, 56.71, 56.59, 56.17, 55.56. IR (KBr) 3398,

2929, 2828, 1705, 1634, 1599, 1513, 1489, 1465, 1438, 1408, 1373,

1319, 1249, 1211, 1108, 1032, 984, 969, 949, 856, 835, 789, 754, 592,

562, 460 cm^ꢀ1; HRMS (þESI) 407.1464 [M þ Na]^þ(calcd. for

tography

(eluent

with

petroleum

ether/ethyl

acetate,

C

₂₂H₂₄O₆Na^þ407.1465).

10:1e5:1e2:1) to afford pure 2,3,4,6-tetramethoxybenzaldehyde

(9.0 g) as a white solid. Yield: 87.4%. Mp 86.0e86.3 ^ꢁC (lit [39]. mp

88.5e89 ^ꢁC). TLC: R_f¼ 0.35 (1:3 EtOAc/hexanes). ¹H NMR (600 MHz,

5.1.24.2. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(3-

methoxyphenyl)acrylate (56). Light yellow solid. Yield: 91.3%. Mp

65.0e65.2 ^ꢁC. TLC: R_f¼ 0.60 (1:1 EtOAc/hexanes). HPLC (purity

Chloroform-d) d 10.29 (s, 1H), 6.25 (s, 1H), 3.94 (s, 3H), 3.93 (s, 3H),

3.89 (s, 3H), 3.80 (s, 3H). ¹³C NMR (151 MHz, Chloroform-d)

d

188.13,

96.6%): t_R¼ 8.24 min ¹H NMR (600 MHz, Chloroform-d)

d 7.69 (d,

159.30, 158.99, 156.86, 136.02, 112.68, 91.68, 62.23, 61.33, 56.32,

56.21. HRMS (þESI) 227.0914 [M þ H]^þ, 249.0732 [M þ Na]^þ(calcd.

for C₁₁H₁₅O^þ₅227.0914 and C₁₁H₁₄O₅Na^þ249.0733).

J ¼ 16.0 Hz, 1H), 7.29 (t, J ¼ 7.9 Hz, 1H), 7.11 (d, J ¼ 7.6 Hz, 1H), 7.04 (s,

1H), 7.00 (s, 1H), 6.99 (d, J ¼ 16.0 Hz, 1H), 6.93 (dd, J ¼ 8.2, 1.7 Hz,

1H), 6.50 (s, 1H), 6.47 (d, J ¼ 16.0 Hz,1H), 6.30e6.22 (m, 1H), 4.87 (d,

J ¼ 6.7 Hz, 2H), 3.89 (s, 3H), 3.86 (s, 3H), 3.83 (s, 3H), 3.81 (s, 3H). ¹³

C

5.1.22. 1,2,4,5-Tetramethoxybenzene (53)

NMR (151 MHz, Chloroform-d) d166.81, 159.92, 151.75, 149.99,

Following the three steps similar to that described for the

preparation of compound 45 except that an equivalent amount of

2,4,5-trimethoxybenzaldehyde 52 (58.9 g, 0.3 mol) was used in

place of 2,3,4-trimethoxybenzaldehyde 44, the title compound 53

was obtained as a white solid (38.8 g). Total yield: 65.2%. Mp

100.4e100.7 ^ꢁC (lit [66]. mp 99.7e101.8 ^ꢁC). TLC: R_f¼ 0.50 (1:1

144.87, 143.34, 135.82, 129.93, 129.22, 121.38, 120.82, 118.43, 116.93,

116.20, 112.97, 110.07, 97.52, 66.04, 56.61, 56.53, 56.10, 55.32. IR

(KBr) 3404, 3048, 2991, 2945, 2836, 1707, 1642, 1593, 1517, 1492,

1468,1452,1436,1408,1311,1292,1252,1224,1212,1175,1037,1010,

990, 969, 927, 853, 839, 784, 677 cm^ꢀ1. HRMS (þESI) 407.1465 [M þ

Na]^þ(calcd. for C₂₂H₂₄O₆Na^þ407.1465).

EtOAc/hexanes). ¹H NMR (600 MHz) 6.60 (s, 2H), 3.84 (s, 12H). ¹³

d C

NMR (151 MHz, Chloroform-d)

d

143.25, 100.76, 57.14. HRMS (þESI)

5.1.24.3. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(4-

methoxyphenyl)acrylate (57). White solid. Yield: 90.6%. Mp

95.5e95.8 ^ꢁC. TLC: R_f¼ 0.55 (1:1 EtOAc/hexanes). HPLC (purity

199.0963 [M þ H]^þ, 221.0786 [M þ Na]^þ(calcd. for C₁₀H₁₅O₄^þ

199.0965 and C₁₀H₁₄O₄Na^þ221.0784).

99.6%): t_R¼ 9.25 min ¹H NMR (600 MHz, Chloroform-d)

d 7.99 (d,

5.1.23. 2,3,5,6-Tetramethoxybenzaldehyde (54)

Following a procedure similar to that described for the prepa-

ration of compound 46 except that an equivalent amount of 1,2,4,5-

J ¼ 16.1 Hz, 1H), 6.98 (t, J ¼ 10.4 Hz, 3H), 6.48 (d, J ¼ 2.3 Hz, 2H), 6.41

(d, J ¼ 16.0 Hz, 1H), 6.26 (dt, J ¼ 15.9, 6.7 Hz, 1H), 4.85 (d, J ¼ 6.6 Hz,

2H), 3.91 (s, 3H), 3.89 (s, 3H), 3.86 (s, 3H), 3.86 (s, 3H), 3.84 (s, 3H),

20

Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

3.83 (s, 3H). ¹³C NMR (151 MHz, Chloroform-d)

d

167.31, 161.50,

56.54, 56.12, 55.81. IR (KBr) 3445, 1702, 1619, 1596, 1518, 1478, 1439,

1406, 1311, 1283, 1258, 1213, 1184, 1158, 1111, 1028, 993, 976, 873,

827, 783, 743 cm^ꢀ1; HRMS (þESI) 437.1571 [M þ Na]^þ(calcd. for

151.80, 150.01, 144.71, 143.43, 129.86, 129.11, 127.30, 121.69, 117.10,

115.68, 114.45, 110.11, 97.63, 65.94, 56.74, 56.61, 56.20, 55.51. IR

(KBr) 2932, 2837, 1705, 1640, 1604, 1577, 1514, 1456, 1425, 1404,

1327, 1254, 1162, 1029, 983, 923, 873, 825, 767, 749, 656, 555, 517,

C

₂₃H₂₆O₇Na^þ437.1571).

474 cm^ꢀ1

₂₂H₂₄O₆Na^þ407.1465).

;

HRMS (þESI) 407.1465 [M

þ

Na]^þ(calcd. for

5.1.24.8. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(3,4-

dimethoxyphenyl)acrylate (62). Gray-yellow solid. Yield: 84.2%.

Mp 79.4e79.8 ^ꢁC. TLC: R_f¼ 0.62 (1:1 EtOAc/hexanes). HPLC (purity

C

5.1.24.4. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(2,3-

dimethoxyphenyl)acrylate (58). White solid. Yield: 90.6%. Mp

81.0e81.3 ^ꢁC. TLC: R_f¼ 0.60 (1:1 EtOAc/hexanes). HPLC (purity

96.7%): t_R¼ 7.04 min ¹H NMR (600 MHz, Chloroform-d)

d 7.66 (d,

J ¼ 15.9 Hz, 1H), 7.10 (d, J ¼ 8.2 Hz, 1H), 7.05 (s, 1H), 6.99 (s, 1H), 6.98

(d, J ¼ 15.4 Hz, 1H), 6.85 (d, J ¼ 8.3 Hz, 1H), 6.49 (s, 1H), 6.35 (d,

J ¼ 15.9 Hz,1H), 6.25 (dt, J ¼ 15.9, 6.7 Hz,1H), 4.85 (d, J ¼ 6.6 Hz, 2H),

3.92e3.87 (m, 9H), 3.85 (s, 3H), 3.83 (s, 3H). ¹³C NMR (151 MHz,

96.3%): t_R¼ 9.25 min ¹H NMR (600 MHz, Chloroform-d)

d 8.02 (d,

J ¼ 16.2 Hz, 1H), 7.14 (d, J ¼ 7.9 Hz, 1H), 7.04 (t, J ¼ 8.0 Hz, 1H), 6.99

(d, J ¼ 15.5 Hz, 1H), 6.99 (s, 1H), 6.93 (d, J ¼ 8.1 Hz, 1H), 6.52 (d,

J ¼ 16.2 Hz, 1H), 6.49 (s, 1H), 6.26 (dt, J ¼ 15.8, 6.7 Hz, 1H), 4.87 (d,

Chloroform-d)

d 167.15, 151.77, 151.20, 150.00, 149.27, 144.92,

143.38, 129.17, 127.49, 122.72, 121.57, 117.02, 115.86, 111.11, 110.09,

109.67, 97.58, 65.94, 56.69, 56.58, 56.15, 56.06, 55.96. IR (KBr) 3452,

2937, 2836,1711,1637, 1600,1518, 1454, 1403, 1337, 1315, 1257, 1232,

1208, 1173, 1152, 1135, 1034, 1022, 993, 978, 869, 845, 810, 798, 751,

J ¼ 6.6 Hz, 2H), 3.89 (s, 3H), 3.86 (d, J ¼ 2.4 Hz, 9H), 3.83 (s, 3H). ¹³

C

NMR (151 MHz, Chloroform-d)

d 167.11, 153.20, 151.75, 149.97,

148.53, 143.37, 139.73, 129.07, 128.69, 124.26, 121.59, 119.50, 119.35,

117.03,114.01, 110.07, 97.57, 66.01, 61.41, 56.67, 56.57, 56.14, 55.94. IR

(KBr) 3449, 2935, 2836, 1715, 1639, 1608, 1581, 1520, 1466, 1404,

1309, 1270, 1218, 1180, 1092, 1066, 1033, 989, 943, 873, 817, 783,

751 cm^ꢀ1; HRMS (þESI) 415.1705 [M þ H]^þ, 437.1571 [M þ Na]^þ

(calcd. for C₂₃H₂₇O₇^þ415.1751 and C₂₃H₂₆O₇Na^þ437.1571).

573 cm^ꢀ1

₂₃H₂₆O₇Na^þ437.1571).

;

HRMS (þESI) 437.1572 [M

þ

Na]^þ(calcd. for

C

5.1.24.9. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(3,5-

dimethoxyphenyl)acrylate (63). White solid. Yield: 80.8%. Mp

133.3e133.5 ^ꢁC. TLC: R_f¼ 0.65 (1:1 EtOAc/hexanes). HPLC (purity

5.1.24.5. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(2,4-

dimethoxyphenyl)acrylate (59). White solid. Yield: 75.3%. Mp

120.0e120.3 ^ꢁC. TLC: R_f¼ 0.52 (1:1 EtOAc/hexanes). HPLC (purity

97.9%): t_R¼ 9.73 min ¹H NMR (600 MHz, Chloroform-d)

d 7.64 (d,

J ¼ 15.9 Hz, 1H), 6.99 (d, J ¼ 15.9 Hz, 1H), 6.99 (s, 1H), 6.66 (d,

J ¼ 2.0 Hz, 2H), 6.52e6.47 (m, 2H), 6.44 (d, J ¼ 15.9 Hz, 1H), 6.25 (dt,

J ¼ 15.9, 6.7 Hz, 1H), 4.86 (d, J ¼ 6.7 Hz, 2H), 3.90 (s, 3H), 3.86 (s, 3H),

3.83 (s, 3H), 3.80 (s, 6H). ¹³C NMR (151 MHz, Chloroform-d)

96.3%): t_R¼ 10.25 min ¹H NMR (600 MHz, Chloroform-d)

d 7.92 (s,

1H), 7.43 (d, J ¼ 8.6 Hz, 1H), 6.98 (d, J ¼ 18.7 Hz, 2H), 6.51e6.43 (m,

4H), 6.26 (dt, J ¼ 15.9, 6.7 Hz, 1H), 4.85 (d, J ¼ 6.6 Hz, 2H), 3.89 (s,

3H), 3.86 (s, 3H), 3.86 (s, 3H), 3.83 (s, 3H), 3.83 (s, 3H). ¹³C NMR

d

166.84, 161.11, 151.81, 150.05, 145.01, 143.41, 136.41, 129.32, 121.41,

118.73, 116.99, 110.11, 106.06, 102.69, 97.59, 66.12, 56.70, 56.60,

56.18, 55.53. IR (KBr) 3448, 2996, 2946, 2838, 1698, 1634, 1599,

1521,1467,1441,1428, 1404,1359,1326, 1293,1276,1241,1214,1166,

1127, 1109, 1068, 1058, 1031, 1012, 997, 972, 919, 858, 835, 752, 674,

(151 MHz, Chloroform-d)

d 167.89, 162.81, 159.99, 151.75, 149.93,

143.41, 140.42, 130.61, 128.89, 121.96, 117.19, 116.72, 116.06, 110.07,

105.32, 98.52, 97.64, 65.78, 56.74, 56.59, 56.18, 55.58. IR (KBr) 3399,

3000, 2949, 2887, 2826, 1708, 1625, 1600, 1518, 1468, 1439, 1421,

1407, 1305, 1282, 1211, 1163, 1107, 1033, 1002, 973, 938, 876, 842,

817, 600, 535, 447 cm^ꢀ1. HRMS (þESI) 437.1571 [M þ Na]^þ(calcd.

for C₂₃H₂₆O₇Na^þ437.1571).

650 cm^ꢀ1

₂₃H₂₆O₇Na^þ437.1571).

;

HRMS (þESI) 437.1572 [M

þ

Na]^þ(calcd. for

C

5.1.24.10. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(2,3,4-

trimethoxyphenyl)acrylate (64). White solid. Yield: 80.8%. Mp

75.2e75.5 ^ꢁC. TLC: R_f¼ 0.56 (1:1 EtOAc/hexanes). HPLC (purity

5.1.24.6. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(2,5-

dimethoxyphenyl)acrylate (60). White solid. Yield: 87.6%. Mp

114.1e114.5 ^ꢁC. TLC: R_f¼ 0.58 (1:1 EtOAc/hexanes). HPLC (purity

99.1%): t_R¼ 7.67 min ¹H NMR (600 MHz, Chloroform-d)

d 7.91 (d,

J ¼ 16.1 Hz, 1H), 7.26 (d, J ¼ 8.8 Hz, 1H), 6.98 (d, J ¼ 15.4 Hz, 2H), 6.69

(d, J ¼ 8.8 Hz, 1H), 6.50 (s, 1H), 6.46 (d, J ¼ 16.1 Hz, 1H), 6.26 (dt,

J ¼ 6.7, 16.0 Hz, 1H), 4.86 (d, J ¼ 6.7 Hz, 2H), 3.92 (s, 3H), 3.90 (s, 3H),

3.89 (s, 3H), 3.87 (s, 3H), 3.87 (s, 3H), 3.84 (s, 3H). ¹³C NMR

97.9%): t_R¼ 8.84 min ¹H NMR (600 MHz, Chloroform-d)

d 8.00 (d,

J ¼ 16.1 Hz, 1H), 7.04 (d, J ¼ 3.0 Hz, 1H), 6.98 (d, J ¼ 13.6 Hz, 2H), 6.89

(dd, J ¼ 9.0, 3.0 Hz, 1H), 6.83 (d, J ¼ 9.0 Hz, 1H), 6.53 (d, J ¼ 16.1 Hz,

1H), 6.49 (s, 1H), 6.26 (dt, J ¼ 15.9, 6.7 Hz, 1H), 4.86 (dd, J ¼ 6.7,

1.0 Hz, 2H), 3.89 (s, 3H), 3.86 (s, 3H), 3.83 (s, 3H), 3.83 (s, 3H), 3.77

(151 MHz, Chloroform-d)

d 167.47, 155.61, 153.41, 151.75, 149.96,

143.40, 142.44, 139.97, 128.96, 123.32, 121.77, 121.60, 117.11, 117.07,

110.08, 107.68, 97.60, 65.88, 61.53, 60.99, 56.70, 56.58, 56.16. IR

(KBr) 2948, 1715, 1639, 1611, 1520, 1469, 1404, 1317, 1209, 1182, 1110,

1032, 979, 870, 852, 819, 770, 652, 593, 464 cm^ꢀ1; HRMS (þESI)

467.1676 [M þ Na]^þ(calcd. for C₂₄H₂₈O₈Na^þ467.1676).

(s, 3H). ¹³C NMR (151 MHz, Chloroform-d)

d 167.31, 153.55, 152.92,

151.75, 149.96, 143.38, 140.19, 129.08, 124.02, 121.67, 118.87, 117.21,

117.06, 113.35, 112.51, 110.06, 97.58, 65.96, 56.69, 56.57, 56.14, 55.85.

IR (KBr) 3426, 2999, 2961, 2935, 2830, 1698, 1655, 1629, 1609, 1579,

1502, 1463, 1404, 1376, 1256, 1168, 1109, 1041, 969, 856, 834, 808,

745, 714, 640, 574 cm^ꢀ1. HRMS (þESI) 437.1571 [M þ Na]^þ(calcd. for

5.1.24.11. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(2,3,6-

trimethoxyphenyl)acrylate (65). Light yellow solid. Yield: 76.4%.

Mp 91.9e92.3 ^ꢁC. TLC: R_f¼ 0.50 (1:1 EtOAc/hexanes). HPLC (purity

C

₂₃H₂₆O₇Na^þ437.1571).

5.1.24.7. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(2,6-

dimethoxyphenyl)acrylate (61). White solid. Yield: 70.3%. Mp

121.9e122.3 ^ꢁC. TLC: R_f¼ 0.62 (1:1 EtOAc/hexanes). HPLC (purity

98.5%): t_R¼ 7.75 min ¹H NMR (600 MHz, Chloroform-d)

d 8.05 (d,

J ¼ 16.3 Hz, 1H), 7.00 (d, J ¼ 3.5 Hz, 2H), 6.97 (d, J ¼ 3.8 Hz, 1H), 6.88

(d, J ¼ 9.0 Hz, 1H), 6.59 (d, J ¼ 9.1 Hz, 1H), 6.50 (s, 1H), 6.27 (dt,

J ¼ 6.7,16.0 Hz,1H), 4.87 (dd, J ¼ 0.6, 6.7 Hz, 2H), 3.89 (s, 3H), 3.86 (s,

3H), 3.84 (s, 3H), 3.83 (s, 3H), 3.83 (s, 6H). ¹³C NMR (151 MHz,

97.1%): t_R¼ 9.49 min ¹H NMR (600 MHz, Chloroform-d)

d 8.18 (d,

J ¼ 16.3 Hz, 1H), 7.25 (t, J ¼ 8.5 Hz, 1H), 6.98 (d, J ¼ 17.8 Hz, 2H), 6.93

(d, J ¼ 16.3 Hz, 1H), 6.54 (d, J ¼ 8.4 Hz, 2H), 6.49 (s, 1H), 6.28 (dt,

J ¼ 15.9, 6.7 Hz, 1H), 4.87 (d, J ¼ 6.7 Hz, 2H), 3.89 (s, 3H), 3.86 (d,

J ¼ 1.1 Hz, 9H), 3.83 (s, 3H). ¹³C NMR (151 MHz, Chloroform-d)

Chloroform-d)

d 168.27, 153.76, 151.75, 149.94, 147.11, 143.42,

136.02, 128.90, 122.11, 121.96, 118.18, 117.21, 114.55, 110.08, 105.86,

97.65, 65.89, 61.03, 56.74, 56.61, 56.60, 56.18, 55.99. IR (KBr) 3396,

3043, 3002, 2964, 2935, 2838, 1706, 1633, 1608, 1582, 1515, 1486,

1466,1406,1325,1256,1201,1159,1104,1068,1042,1028,1008, 995,

d

168.55, 160.10, 151.69, 149.86, 143.35, 135.80, 131.30, 128.78,

122.06, 120.54, 117.19, 112.31, 110.03, 103.71, 97.60, 65.72, 56.69,

Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

21

968, 941, 874, 853, 794, 722 cm^ꢀ1. HRMS (þESI) 467.1676 [M þ Na]^þ

121.3e121.6 ^ꢁC. TLC: R_f¼ 0.50 (1:1 EtOAc/hexanes). HPLC (purity

(calcd. for C₂₄H₂₈O₈Na^þ467.1676).

99.5%): t_R¼ 8.58 min ¹H NMR (600 MHz, Chloroform-d)

d 8.01 (d,

J ¼ 16.3 Hz, 1H), 7.00 (s, 1H), 6.98 (d, J ¼ 16.0 Hz, 1H), 6.84 (d,

J ¼ 16.3 Hz, 1H), 6.49 (s, 1H), 6.30e6.24 (m, 2H), 4.85 (dd, J ¼ 6.7,

0.9 Hz, 2H), 3.90 (s, 6H), 3.89 (s, 3H), 3.87 (s, 3H), 3.86 (s, 3H), 3.83

5.1.24.12. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(2,4,5-

trimethoxyphenyl)acrylate (66). Light yellow solid. Yield: 86.8%.

Mp 114.8e115.2 ^ꢁC. TLC: R_f¼ 0.60 (1:1 EtOAc/hexanes). HPLC (pu-

(s, 3H), 3.80 (s, 3H). ¹³C NMR (151 MHz, Chloroform-d)

d 168.68,

rity 95.9%): t_R¼ 6.94 min ¹H NMR (600 MHz, Chloroform-d)

d

7.99

156.42, 155.43, 154.50, 151.74, 149.90, 143.43, 136.43, 135.97, 128.73,

122.17, 118.94, 117.28, 110.41, 110.08, 97.67, 91.97, 65.72, 61.31, 61.19,

56.76, 56.60, 56.19, 56.14, 55.97. IR (KBr) 3439, 2996, 2937, 2838,

1699, 1623, 1600, 1572, 1498, 1463, 1439, 1404, 1344, 1299, 1288,

1237, 1209, 1157, 1110, 1043, 1030, 1015, 983, 923, 864, 827,

810 cm^ꢀ1. HRMS (þESI) 497.1783 [M þ Na]^þ(calcd. for C₂₅H₃₀O₉Na^þ

497.1782).

(d, J ¼ 16.1 Hz, 1H), 7.02e6.96 (m, 3H), 6.48 (d, J ¼ 2.3 Hz, 2H), 6.41

(d, J ¼ 16.0 Hz, 1H), 6.26 (dt, J ¼ 6.7, 15.9 Hz, 1H), 4.85 (d, J ¼ 6.6 Hz,

2H), 3.91 (s, 3H), 3.89 (s, 3H), 3.86 (s, 3H), 3.86 (s, 3H), 3.84 (s, 3H),

3.83 (s, 3H). ¹³C NMR (151 MHz, Chloroform-d)

d 167.69, 153.95,

152.11, 151.67, 149.86, 143.29, 143.22, 139.89, 128.95, 121.77, 116.99,

115.66, 114.92, 110.78, 109.87, 97.43, 96.79, 65.82, 56.67, 56.51,

56.44, 56.39, 56.13. IR (KBr) 3409, 2935, 2830, 1699, 1609, 1515,

1468, 1445, 1406, 1301, 1277, 1209, 1167, 1127, 1031, 998, 969, 869,

809, 541 cm^ꢀ1. HRMS (þESI) 467.1676 [M þ Na]^þ(calcd. for

5.1.24.17. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(2,3,5,6-

tetramethoxyphenyl)acrylate (71). Light yellow solid. Yield: 73.2%.

Mp 117.2e117.6 ^ꢁC. TLC: R_f¼ 0.52 (1:1 EtOAc/hexanes). HPLC (purity

C

₂₄H₂₈O₈Na^þ467.1676).

100.0%): t_R¼ 8.33 min ¹H NMR (600 MHz, Chloroform-d)

d 7.96 (d,

5.1.24.13. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(2,4,6-

trimethoxyphenyl)acrylate (67). White solid. Yield: 57.5%. Mp

150.2e150.5 ^ꢁC. TLC: R_f¼ 0.72 (1:1 EtOAc/hexanes). HPLC (purity

J ¼ 16.3 Hz, 1H), 7.01 (d, J ¼ 16.3 Hz, 1H), 7.00 (s, 1H), 7.99 (d,

J ¼ 16.3 Hz, 1H), 6.59 (s, 1H), 6.49 (s, 1H), 6.27 (dt, J ¼ 6.7, 15.9 Hz,

1H), 4.87 (d, J ¼ 6.2 Hz, 2H), 3.90 (s, 3H), 3.87 (s, 3H), 3.86 (s, 6H),

3.83 (s, 3H), 3.76 (s, 6H). ¹³C NMR (151 MHz, Chloroform-d)

98.8%): t_R¼ 8.14 min ¹H NMR (600 MHz, Chloroform-d)

d 8.12 (d,

J ¼ 16.2 Hz, 1H), 6.99 (s, 1H), 6.96 (d, J ¼ 16.0 Hz, 1H), 6.79 (d,

J ¼ 16.2 Hz, 1H), 6.48 (s, 1H), 6.27 (dt, J ¼ 15.9, 6.7 Hz, 1H), 6.09 (s,

2H), 4.84 (d, J ¼ 6.7 Hz, 2H), 3.88 (s, 3H), 3.85 (s, 3H), 3.85 (s, 6H),

3.82 (s, 3H), 3.82 (s, 3H). ¹³C NMR (151 MHz, Chloroform-d)

d 168.02,151.67, 149.86,149.17, 143.29,142.32,136.17,128.94,122.97,

122.69, 121.72, 116.96, 109.82, 97.38, 66.04, 60.98, 56.68, 56.58,

56.52, 56.13. IR (KBr) 3435, 2935, 2835, 1711, 1628, 1587, 1517, 1487,

1467, 1404,1349, 1290,1245, 1209,1167,1095,1063, 1033, 1009, 972,

873, 821 cm^ꢀ1. HRMS (þESI) 497.1783 [M þ Na]^þ(calcd. for

d

168.98, 162.86, 161.36, 151.68, 149.82, 143.37, 135.87, 128.63,

122.29, 117.40, 117.28, 110.03, 105.93, 97.64, 90.47, 65.57, 56.72,

56.55, 56.14, 55.77, 55.45. IR (KBr) 3442, 2936, 1700, 1604, 1519,

1493,1468, 1439,1404,1309,1288,1233,1214,1159, 1123,1032, 995,

973, 954, 875, 811, 524 cm^ꢀ1; HRMS (þESI) 467.1676 [M þ Na]^þ

(calcd. for C₂₄H₂₈O₈Na^þ467.1676).

C

₂₅H₃₀O₉Na^þ497.1782).

5.1.24.18. (E)-3-(2,4,5-trimethoxyphenyl)allyl(E)-3-(2,3,4,5,6-

pentamethoxyphenyl) acrylate (72). White solid. Yield: 64.5%. Mp

102.0e102.5 ^ꢁC. TLC: R_f¼ 0.67 (1:1 EtOAc/hexanes). HPLC (purity

99.1%): t_R¼ 10.15. ¹H NMR (600 MHz, Chloroform-d)

d 7.90 (d,

5.1.24.14. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(3,4,5-

trimethoxyphenyl)acrylate (68). Light yellow solid. Yield: 71.2%.

Mp 72.0e72.3 ^ꢁC. TLC: R_f¼ 0.62 (1:1 EtOAc/hexanes). HPLC (purity

J ¼ 16.3 Hz, 1H), 6.99 (s, 1H), 6.98 (d, J ¼ 18.0 Hz, 1H), 6.91 (d,

J ¼ 16.3 Hz, 1H), 6.49 (s, 1H), 6.26 (dt, J ¼ 6.7, 15.9 Hz, 1H), 4.86 (d,

J ¼ 6.6 Hz, 2H), 3.98 (s, 3H), 3.89 (s, 3H), 3.86 (s, 3H), 3.86 (s, 6H),

95.1%): t_R¼ 6.66 min ¹H NMR (600 MHz, Chloroform-d)

d

7.64 (d,

3.84 (s, 6H), 3.83 (s, 3H). ¹³C NMR (151 MHz, Chloroform-d)

d 151.65,

J ¼ 15.9 Hz, 1H), 7.00 (d, J ¼ 15.9 Hz, 1H), 7.00 (s, 1H), 6.76 (s, 2H),

6.51 (s, 1H), 6.39 (d, J ¼ 15.9 Hz, 1H), 6.26 (dt, J ¼ 15.9, 6.7 Hz, 1H),

4.87 (d, J ¼ 6.7 Hz, 2H), 3.91 (s, 3H), 3.88 (s, 6H), 3.88 (s, 3H), 3.87 (s,

149.84, 149.58, 149.36, 143.27, 143.24, 135.83, 128.83, 121.80, 121.03,

117.37, 116.97, 109.81, 97.37, 65.92, 65.90, 61.60, 61.38, 61.22, 56.66,

56.51, 56.12. IR (KBr) 3400, 2974, 2941, 2839, 1705, 1624, 1589, 1513,

1467, 1408, 1323, 1301, 1267, 1211, 1171, 1126, 1062, 1034, 1001, 976,

957, 937, 868 cm^ꢀ1. HRMS (þESI) 527.1889 [M þ Na]^þ(calcd. for

3H), 3.85 (s, 3H). ¹³C NMR (151 MHz, Chloroform-d)

d 166.93,

153.56, 151.84, 150.10, 145.02, 143.44, 140.22, 130.07, 129.38, 121.44,

117.48, 117.01, 110.15, 105.33, 97.61, 66.12, 61.12, 56.74, 56.65, 56.28,

56.21. IR (KBr) 3513, 2925, 1709, 1636, 1583, 1508, 1464, 1245, 1128,

1031, 976, 835, 632 cm^ꢀ1; HRMS (þESI) 467.1678 [M þ Na]^þ(calcd.

for C₂₄H₂₈O₈Na^þ467.1676).

C

₂₆H₃₂O₁₀Na^þ527.1888).

5.1.24.19. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(2-ﬂuorophenyl)

acrylate (73). Light yellow solid. Yield: 72.9%. Mp 73.5e73.8 ^ꢁC.

TLC: R_f¼ 0.50 (1:1 EtOAc/hexanes). HPLC (purity 100.0%):

5.1.24.15. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(2,3,4,5-

tetramethoxyphenyl)acrylate (69). Light yellow solid. Yield: 70.4%.

Mp 68.3e69.7 ^ꢁC. TLC: R_f¼ 0.73 (1:1 EtOAc/hexanes). HPLC (purity

t_R¼ 9.17 min ¹H NMR (600 MHz, Chloroform-d)

d 7.85 (d,

J ¼ 16.2 Hz, 1H), 7.54 (t, J ¼ 7.5 Hz, 1H), 7.38e7.32 (m, 1H), 7.16 (t,

J ¼ 7.5 Hz, 1H), 7.13e7.06 (m, 1H), 7.00 (d, J ¼ 15.9 Hz, 1H), 7.00 (s,

1H) 6.58 (d, J ¼ 16.2 Hz, 1H), 6.50 (s, 1H), 6.26 (dt, J ¼ 15.9, 6.7 Hz,

99.3%): t_R¼ 9.06 min ¹H NMR (600 MHz, Chloroform-d)

d 7.95 (d,

J ¼ 16.1 Hz, 1H), 7.00 (s, 1H), 6.99 (d, J ¼ 15.9 Hz, 1H), 6.79 (s, 1H),

6.50 (s, 1H), 6.44 (d, J ¼ 16.1 Hz, 1H), 6.26 (dt, J ¼ 15.9, 6.7 Hz, 1H),

4.87 (dd, J ¼ 6.7, 0.8 Hz, 2H), 3.93 (s, 3H), 3.92 (s, 3H), 3.90 (s, 3H),

3.86 (s, 3H), 3.85 (s, 3H), 3.85 (s, 3H), 3.84 (s, 3H). ¹³C NMR

1H), 4.88 (d, J ¼ 6.7 Hz, 2H), 3.90 (s, 3H), 3.87 (s, 3H), 3.84 (s, 3H). ¹³

C

NMR (151 MHz, Chloroform-d)

d 166.74, 162.27, 160.58, 151.79,

150.02, 143.38, 137.56, 131.75, 129.29, 124.51, 122.53, 121.35, 120.75,

116.96,116.20, 110.08, 97.56, 66.17, 56.66, 56.57, 56.14. IR (KBr) 3416,

1709, 1640, 1608, 1513, 1466, 1403, 1318, 1276, 1212, 1030, 974, 944,

860, 839, 805, 767, 651, 460, 411 cm^ꢀ1; HRMS (þESI) 395.1265 [M þ

Na]^þ(calcd. for C₂₁H₂₁FO₅Na^þ395.1265).

(151 MHz, Chloroform-d)

d 167.18, 151.81, 150.04, 149.78, 147.75,

147.34, 145.40, 143.43, 139.58, 129.17, 122.80, 121.62, 118.09, 117.08,

110.12, 104.52, 97.63, 66.04, 61.98, 61.41, 61.33, 56.73, 56.63, 56.28,

56.20. IR (KBr) 3426, 3056, 2996, 2943, 2833,1709,1628,1606,1591,

1520,1490,1468,1435,1414,1353,1322,1287,1247,1207,1165,1132,

1088, 1038, 1008, 990, 970, 934, 884, 861, 846, 814 cm^ꢀ1. HRMS

(þESI) 497.1783 [M þ Na]^þ(calcd. for C₂₅H₃₀O₉Na^þ497.1782).

5.1.24.20. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(3-ﬂuorophenyl)

acrylate (74). White solid. Yield: 75.4%. Mp 89.9e90.2 ^ꢁC. TLC:

R_f¼ 0.66 (1:1 EtOAc/hexanes). HPLC (purity 100.0%): t_R¼ 10.41 min

¹H NMR (600 MHz, Chloroform-d)

d

7.67 (d, J ¼ 16.0 Hz, 1H),

5.1.24.16. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(2,3,4,6-

tetramethoxyphenyl)acrylate (70). White solid. Yield: 63.5%. Mp

7.39e7.31 (m, 1H), 7.29 (d, J ¼ 7.7 Hz, 1H), 7.21 (d, J ¼ 9.6 Hz, 1H),

7.07 (td, J ¼ 8.3,1.7 Hz,1H), 7.00 (d, J ¼ 15.9 Hz,1H), 6.99 (s,1H), 6.50

22

Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

(s, 1H), 6.47 (d, J ¼ 16.0 Hz, 1H), 6.25 (dt, J ¼ 15.9, 6.7 Hz, 1H), 4.87

(dd, J ¼ 6.7, 0.8 Hz, 2H), 3.90 (s, 3H), 3.87 (s, 3H), 3.84 (s, 3H). ¹³C

1111, 1033, 976, 937, 868, 847, 823, 810, 746, 650, 501, 476 cm^ꢀ1

;

HRMS (þESI) 411.0972 [M þ Na]^þ(calcd. for C₂₁H₂₁ClO₅Na^þ

NMR (151 MHz, Chloroform-d)

d

166.53, 163.89, 162.25, 151.81,

411.0970).

150.07, 143.55, 143.39, 136.78, 130.56, 129.40, 124.14, 121.26, 119.61,

117.15, 114.32, 110.11, 97.55, 66.22, 56.65, 56.58, 56.15. IR (KBr) 1711,

1643, 1612, 1519, 1440, 1405, 1337, 1272, 1211, 1168, 1119, 1079, 1029,

970, 861, 824, 803, 728, 694, 657 cm^ꢀ1; HRMS (þESI) 395.1266 [M þ

Na]^þ(calcd. for C₂₁H₂₁FO₅Na^þ395.1265).

5.1.24.25. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(2-

bromophenyl)acrylate (79). Light yellow solid. Yield: 79.3%. Mp

87.3e87.6 ^ꢁC. TLC: R_f¼ 0.60 (1:1 EtOAc/hexanes). HPLC (purity

97.2%): t_R¼ 9.03 min ¹H NMR (600 MHz, Chloroform-d)

d 8.09 (d,

J ¼ 15.9 Hz, 1H), 7.60 (ddd, J ¼ 7.6, 4.3, 1.0 Hz, 2H), 7.32 (t, J ¼ 7.5 Hz,

1H), 7.22 (t, J ¼ 7.7 Hz, 1H), 7.00 (d, J ¼ 15.9 Hz, 1H), 6.99 (s, 1H), 6.50

(s,1H), 6.43 (d, J ¼ 15.9 Hz,1H), 6.26 (dt, J ¼ 15.9, 6.7 Hz,1H), 4.88 (d,

J ¼ 6.7 Hz, 2H), 3.90 (s, 3H), 3.87 (s, 3H), 3.84 (s, 3H). ¹³C NMR

5.1.24.21. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(4-ﬂuorophenyl)

acrylate (75). White solid. Yield: 71.6%. Mp 92.9e93.3 ^ꢁC. TLC:

R_f¼ 0.55 (1:1 EtOAc/hexanes). HPLC (purity 98.8%): t_R¼ 8.59 min

¹H NMR (600 MHz, Chloroform-d)

d

7.67 (d, J ¼ 16.0 Hz, 1H), 7.50

(151 MHz, Chloroform-d) d166.38, 151.83, 150.07, 143.43, 143.36,

(dd, J ¼ 8.5, 5.5 Hz, 2H), 7.06 (t, J ¼ 8.6 Hz, 2H), 6.99 (s, 1H), 6.98 (d,

J ¼ 16.0 Hz, 1H), 6.49 (s, 1H), 6.39 (d, J ¼ 16.0 Hz, 1H), 6.25 (dt,

J ¼ 15.9, 6.7 Hz,1H), 4.86 (d, J ¼ 6.7 Hz, 2H), 3.89 (s, 3H), 3.86 (s, 3H),

134.64, 133.56, 131.32, 129.30, 127.90, 127.83, 125.46, 121.37, 121.08,

117.01, 110.13, 97.61, 66.26, 56.73, 56.63, 56.20. IR (KBr) 3449, 1712,

1636, 1609, 1513, 1469, 1439, 1407, 1314, 1263, 1208, 1178, 1032, 973,

855, 762 cm^ꢀ1; HRMS (þESI) 455.0464 [M þ Na]^þ(calcd. for

3.83 (s, 3H). ¹³C NMR (151 MHz, Chloroform-d)

d 166.80, 164.82,

163.15, 151.81, 150.06, 143.68, 143.40, 130.77, 130.01, 129.32, 121.39,

117.95, 116.96, 116.22, 116.07, 110.11, 97.57, 66.12, 56.68, 56.59, 56.16.

IR (KBr) 2937, 1705, 1640, 1607, 1590, 1517, 1461, 1401, 1374, 1310,

1174, 1129, 1110, 1033, 975, 937, 867, 823, 810, 746, 650, 501,

C

₂₁H₂₁BrO₅Na^þ455.0465).

5.1.24.26. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(3-

bromophenyl)acrylate (80). White solid. Yield: 86.3%. Mp

77.5e77.8 ^ꢁC. TLC: R_f¼ 0.58 (1:1 EtOAc/hexanes). HPLC (purity

476 cm^ꢀ1

₂₁H₂₁FO₅Na^þ395.1265).

;

HRMS (þESI) 395.1265 [M

þ

Na]^þ(calcd. for

C

98.5%): t_R¼ 9.49 min ¹H NMR (600 MHz, Chloroform-d)

d 7.71 (s,

5.1.24.22. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(2-chlorophenyl)

acrylate (76). Light yellow solid. Yield: 90.3%. Mp 65.2e65.5 ^ꢁC.

TLC: R_f¼ 0.56 (1:1 EtOAc/hexanes). HPLC (purity 99.2%):

1H), 7.68 (d, J ¼ 16.0 Hz, 1H), 7.54 (d, J ¼ 8.0 Hz, 1H), 7.48 (d,

J ¼ 7.8 Hz, 1H), 7.30 (dd, J ¼ 12.7, 4.7 Hz, 1H), 7.04 (d, J ¼ 16.0 Hz, 1H),

7.04 (s, 1H), 6.55 (s, 1H), 6.51 (d, J ¼ 16.0 Hz, 1H), 6.30 (dt, J ¼ 15.9,

6.7 Hz, 1H), 4.92 (d, J ¼ 6.7 Hz, 2H), 3.95 (s, 3H), 3.92 (s, 3H), 3.89 (s,

t_R¼ 10.75 min ¹H NMR (600 MHz, Chloroform-d)

d 8.13 (d,

J ¼ 16.0 Hz, 1H), 7.62 (d, J ¼ 7.5 Hz, 1H), 7.41 (d, J ¼ 7.6 Hz, 1H),

7.34e7.24 (m, 2H), 7.00 (d, J ¼ 15.9 Hz, 1H), 7.0 (s, 1H), 6.50 (s, 1H),

6.47 (d, J ¼ 16.0 Hz, 1H), 6.26 (dt, J ¼ 15.8, 6.7 Hz, 1H), 4.89 (d,

J ¼ 6.7 Hz, 2H), 3.90 (s, 3H), 3.87 (s, 3H), 3.84 (s, 3H). ¹³C NMR

3H). ¹³C NMR (151 MHz, Chloroform-d)

d 166.47, 151.82, 150.08,

143.40, 143.25, 136.63, 133.14, 130.87, 130.50, 129.41, 126.75, 123.12,

121.26, 119.72, 116.93, 110.12, 97.56, 66.24, 56.68, 56.60, 56.17. IR

(KBr) 2927,1720,1638,1611,1559,1518,1473,1404,1377,1296,1209,

1029, 1002, 969, 883, 859, 821, 782, 729, 669, 575, 533 cm^ꢀ1; HRMS

(þESI) 455.0465 [M þ Na]^þ(calcd. for C₂₁H₂₁BrO₅Na^þ455.0465).

(151 MHz, Chloroform-d)

d 166.47, 151.81, 150.05, 143.41, 140.79,

135.06, 132.82, 131.15, 130.28, 129.30, 127.74, 127.19, 121.35, 120.87,

116.99, 110.11, 97.59, 66.25, 56.71, 56.61, 56.18. IR (KBr) 1708, 1636,

1606, 1586, 1513, 1468, 1403, 1369, 1315, 1267, 1230, 1211, 1174, 1135,

1030, 973, 941, 859, 835, 767, 748, 681, 585, 448 cm^ꢀ1; HRMS (þESI)

411.0971 [M þ Na]^þ(calcd. for C₂₁H₂₁ClO₅Na^þ411.0970).

5.1.24.27. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(4-

bromophenyl)acrylate (81). White solid. Yield: 81.6%. Mp

105.3e105.6 ^ꢁC. TLC: R_f¼ 0.56 (1:1 EtOAc/hexanes). HPLC (purity

97.0%): t_R¼ 9.61 min ¹H NMR (600 MHz, Chloroform-d)

d 7.64 (d,

5.1.24.23. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(3-chlorophenyl)

acrylate (77). Light yellow solid. Yield: 82.4%. Mp 78.2e78.4 ^ꢁC.

TLC: R_f¼ 0.60 (1:1 EtOAc/hexanes). HPLC (purity 97.4%):

J ¼ 16.0 Hz, 1H), 7.51 (d, J ¼ 8.4 Hz, 2H), 7.38 (d, J ¼ 8.4 Hz, 2H), 6.99

(d, J ¼ 15.9 Hz, 1H), 6.99 (s, 1H), 6.50 (s, 1H), 6.46 (d, J ¼ 16.0 Hz, 1H),

6.24 (dt, J ¼ 15.9, 6.7 Hz, 1H), 4.86 (d, J ¼ 6.7 Hz, 2H), 3.90 (s, 3H),

t_R¼ 11.66 min ¹H NMR (600 MHz, Chloroform-d)

d 7.64 (d,

3.86 (s, 3H), 3.84 (s, 3H). ¹³C NMR (151 MHz, Chloroform-d)

d166.68,

J ¼ 16.0 Hz, 1H), 7.50 (s, 1H), 7.39 (d, J ¼ 7.4 Hz, 1H), 7.36e7.28 (m,

2H), 6.99 (d, J ¼ 15.9 Hz, 1H), 6.99 (s, 1H), 6.50 (s, 1H), 6.47 (d,

J ¼ 16.0 Hz,1H), 6.25 (dt, J ¼ 15.9, 6.7 Hz,1H), 4.87 (d, J ¼ 6.7 Hz, 2H),

3.90 (s, 3H), 3.87 (s, 3H), 3.84 (s, 3H). ¹³C NMR (151 MHz, Chloro-

151.83, 150.09, 143.62, 143.42, 133.46, 132.26, 129.56, 129.41, 124.65,

121.31, 118.93, 116.95, 110.13, 97.58, 66.22, 56.70, 56.62, 56.19. IR

(KBr) 3437, 2936, 2837, 1705, 1640, 1607, 1586, 1517, 1487, 1462,

1450, 1399, 1311, 1291, 1276, 1213, 1176, 1129, 1110, 1071, 1043, 1033,

1008, 974, 937, 870, 820, 496 cm^ꢀ1; HRMS (þESI) 455.0463 [M þ

Na]^þ(calcd. for C₂₁H₂₁BrO₅Na^þ455.0465).

form-d)

d 166.51, 151.82, 150.08, 143.40, 143.36, 136.35, 135.01,

130.24, 129.41, 127.92, 126.32, 121.27, 119.70, 116.93, 110.12, 97.56,

66.25, 56.68, 56.60, 56.17. IR (KBr) 3430, 2930, 1721, 1639, 1611,

1564, 1519, 1476, 1405, 1378, 1345, 1295, 1272, 1209, 1169, 1124,

1076,1029,1003, 970, 886, 858, 822, 784, 735, 671, 577 cm^ꢀ1; HRMS

(þESI) 411.0972 [M þ Na]^þ(calcd. for C₂₁H₂₁ClO₅Na^þ411.0970).

5.1.24.28. (E)-3-(2,4,5-trimethoxyphenyl)allyl

(E)-3-(2-(tri-

ﬂuoromethyl)phenyl)acrylate (82). White solid. Yield: 74.2%. Mp

84.8e85.1 ^ꢁC. TLC: R_f¼ 0.65 (1:1 EtOAc/hexanes). HPLC (purity

5.1.24.24. (E)-3-(2,4,5-trimethoxyphenyl)allyl (E)-3-(4-chlorophenyl)

acrylate (78). Light yellow solid. Yield: 85.1%. Mp 92.6e92.9 ^ꢁC.

TLC: R_f¼ 0.56 (1:1 EtOAc/hexanes). HPLC (purity 98.1%):

99.0%): t_R¼ 7.77 min ¹H NMR (600 MHz, Chloroform-d)

d 8.10 (dd,

J ¼ 15.8, 1.7 Hz, 1H), 7.69 (d, J ¼ 8.0 Hz, 2H), 7.55 (t, J ¼ 7.6 Hz, 1H),

7.47 (t, J ¼ 7.6 Hz, 1H), 7.00 (d, J ¼ 15.7 Hz, 1H), 6.99 (s, 1H), 6.50 (s,

1H), 6.44 (d, J ¼ 15.8 Hz, 1H), 6.26 (dt, J ¼ 16.0, 6.6 Hz, 1H), 4.88 (dd,

J ¼ 6.7, 1.0 Hz, 2H), 3.89 (s, 3H), 3.86 (s, 3H), 3.83 (s, 3H). ¹³C NMR

t_R¼ 9.27 min ¹H NMR (600 MHz, Chloroform-d)

d 7.66 (d,

J ¼ 16.0 Hz, 1H), 7.44 (d, J ¼ 8.5 Hz, 2H), 7.35 (d, J ¼ 8.5 Hz, 2H), 6.99

(d, J ¼ 16.0 Hz, 1H), 6.99 (s, H), 6.49 (s, 1H), 6.44 (d, J ¼ 16.0 Hz, 1H),

6.29e6.21 (m, 1H), 4.86 (dd, J ¼ 6.7, 1.0 Hz, 2H), 3.89 (s, 3H), 3.86 (s,

(151 MHz, Chloroform-d)

d 166.06, 151.84, 150.07, 143.42, 140.45,

133.52, 132.21, 129.69,129.30,128.02, 126.25, 124.94,123.12,122.58,

121.26,116.98,110.14, 97.60, 66.31, 56.69, 56.61, 56.18. IR (KBr) 2948,

1715, 1639, 1611, 1520, 1469, 1404, 1317, 1209, 1182, 1110, 1032, 979,

870, 852, 819, 770, 652, 593, 464 cm^ꢀ1; HRMS (þESI) 445.1236

[M þ Na]^þ(calcd. for C₂₂H₂₁F₃O₅Na^þ445.1233).

3H), 3.83 (s, 3H). ¹³C NMR (151 MHz, Chloroform-d)

d 166.70,151.83,

150.09,143.56,143.42,136.30,133.03,129.40,129.35,129.30,121.33,

118.81, 116.96,110.13, 97.58, 66.21, 56.70, 56.62, 56.19. IR (KBr) 2937,

2833, 1705, 1640, 1607, 1590, 1517, 1461, 1401, 1374, 1310, 1213, 1130,

Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

23

5.1.24.29. (E)-3-(2,4,5-trimethoxyphenyl)allyl

(E)-3-(3-(tri-

3.88 (s, 3H), 3.87 (s, 6H), 3.86 (s, 6H), 3.84 (s, 3H). ¹³C NMR

(126 MHz, Chloroform-d) 164.75, 153.53, 151.69, 149.40, 143.35,

141.31, 139.72, 137.89, 130.63, 120.37, 120.06, 114.85, 113.43, 105.14,

98.54, 61.08, 56.77, 56.70, 56.39, 56.30, 53.24. IR (KBr) 3361, 3283,

2987, 2939, 2836, 1659, 1618, 1580, 1512, 1462, 1415, 1403, 1325,

1277, 1248, 1209, 1152, 1127, 1034, 1007, 828, 608 cm^ꢀ1. HRMS

(þESI) 444.2000 [M þ H]^þ(calcd. for C₂₄H₃₀NO^þ₇444.2017).

ﬂuoromethyl)phenyl)acrylate (83). White solid. Yield: 77.7%. Mp

d

126.4e126.6 ^ꢁC. TLC: R_f¼ 0.56 (1:1 EtOAc/hexanes). HPLC (purity

100.0%): t_R¼ 10.31 min ¹H NMR (600 MHz, Chloroform-d)

d 7.75 (s,

1H), 7.72 (d, J ¼ 16.0 Hz, 1H), 7.68 (d, J ¼ 7.7 Hz, 1H), 7.62 (d,

J ¼ 7.6 Hz, 1H), 7.50 (t, J ¼ 7.7 Hz, 1H), 6.99 (d, J ¼ 15.7 Hz, 1H), 6.99

(s, 1H), 6.53 (d, J ¼ 16.0 Hz, 1H), 6.50 (s, 1H), 6.25 (dt, J ¼ 6.7, 15.8 Hz,

1H), 4.88 (d, J ¼ 6.6 Hz, 2H), 3.89 (s, 3H), 3.86 (s, 3H), 3.83 (s, 3H). ¹³

C

NMR (151 MHz, Chloroform-d)

d 166.36, 151.83, 150.10, 143.40,

5.1.25.2. (E)-3-(3,4,5-trimethoxyphenyl)-N-(3-(2,4,5-

trimethoxyphenyl)propyl)acrylamide (87). White solid. Yield: 86.6%.

Mp 159.6e159.8 ^ꢁC. TLC: R_f¼ 0.27 (1:1 EtOAc/hexanes). HPLC (pu-

143.13, 135.32, 131.64, 131.13, 129.56, 129.49, 126.73, 124.69, 122.97,

121.17, 120.20, 116.89, 110.12, 97.54, 66.30, 56.65, 56.59, 56.15. IR

(KBr) 2936, 1715, 1643, 1624, 1518, 1492, 1468, 1436, 1402, 1382,

1337, 1163, 1035, 983, 860, 804, 774, 761, 743, 711, 692, 659, 636,

603, 577, 559, 513, 449, 416 cm^ꢀ1; HRMS (þESI) 445.1236 [M þ Na]^þ

(calcd. for C₂₂H₂₄O₆Na^þ445.1233).

rity 100.0%): t_R¼ 6.22 min ¹H NMR (500 MHz, Chloroform-d)

d 7.52

(d, J ¼ 15.5 Hz, 1H), 6.78 (s, 1H), 6.72 (s, 2H), 6.59 (d, J ¼ 9.2 Hz, 1H),

6.55 (s, 1H), 6.29 (d, J ¼ 15.5 Hz, 1H), 5.09e5.02 (m, 1H), 3.89 (s, 3H),

3.88 (s, 3H), 3.87 (s, 6H), 3.86 (s, 3H), 3.84 (s, 3H), 1.93e1.80 (m, 2H),

0.88 (t, J ¼ 7.4 Hz, 3H). ¹³C NMR (126 MHz, Chloroform-d)

d 164.74,

5.1.24.30. (E)-3-(2,4,5-trimethoxyphenyl)allyl

(E)-3-(4-(tri-

153.39, 151.47, 148.75, 142.97, 140.78, 130.62, 121.21, 120.66, 113.64,

104.98, 98.25, 60.95, 56.66, 56.35, 56.27, 56.17, 53.84, 28.63,11.24. IR

(KBr) 3422, 3291, 2957, 2937, 2839, 1658, 1616, 1584, 1510, 1461,

ﬂuoromethyl)phenyl)acrylate (84). Light yellow solid. Yield: 84.8%.

Mp 109.5e109.7 ^ꢁC. TLC: R_f¼ 0.66 (1:1 EtOAc/hexanes). HPLC (pu-

rity 98.6%): t_R¼ 10.38 min ¹H NMR (600 MHz, Chloroform-d)

d 7.72

1421, 1324, 1278, 1240, 1209, 1152, 1128, 1035, 1001, 829, 610 cm^ꢀ1

.

(d, J ¼ 16.0 Hz, 1H), 7.63 (q, J ¼ 8.6 Hz, 4H), 7.00 (d, J ¼ 15.6 Hz, 1H),

6.99 (s, 1H), 6.54 (d, J ¼ 16.0 Hz, 1H), 6.50 (s, 1H), 6.25 (dt, J ¼ 6.8,

15.9 Hz,1H), 4.88 (d, J ¼ 6.7 Hz, 2H), 3.90 (s, 3H), 3.86 (s, 3H), 3.84 (s,

HRMS (þESI) 446.2151 [M þ H]^þ(calcd. for C₂₄H₃₂NO^þ₇446.2173).

3H). ¹³C NMR (151 MHz, Chloroform-d)

d 166.38, 151.87, 150.15,

5.2. Biological evaluation

143.43, 143.12, 137.92, 131.76, 129.59, 128.30, 125.98, 124.84, 123.03,

121.15, 120.82, 116.89, 110.16, 97.57, 66.38, 56.69, 56.63, 56.19. IR

(KBr) 3449, 2943, 2838, 1704, 1612, 1519, 1469, 1448, 1405, 1325,

1289, 1248, 1211, 1157, 1118, 1065, 1048, 1028, 1010, 983, 963, 866,

832, 753, 656, 591, 502, 418 cm^ꢀ1; HRMS (þESI) 445.1235 [M þ

Na]^þ(calcd. for C₂₂H₂₁F₃O₅Na^þ445.1233).

5.2.1. Animals and drugs

All animals experiments were performed with the use of male

Kunming mice (Experimental Animal Center, Xi'an Jiaotong Uni-

versity, Xi'an, China) weighing 25e30 g. All the mice were kept

under controlled environmental conditions (22 2 ^ꢁC; 50 20%

humidity; 12/12 h light/dark cycle) with free access to pellet food

and water. Experiments were performed following approval of

Committee on Ethics of Experimental Animal Administration of the

Northwest University and were in accordance with the National

Institutes of Health guidelines [76]. Each experiment consisted of

several groups of 4e6 mice, one group received the vehicle control

(normal saline (NS) containing 0.5% Tween-80) and the other

groups received different doses of tested compounds. All tested

compounds were dissolved in 0.5% Tween-80 and injected ip to

mice (10 mL/kg volume) 30 min before testing. In scPTZ and sc3-MP

tests, the time-effect relationship was determined at 0.25, 0.5, 1.0

and 2.0 h after drugs treatment. PTZ was purchased from Alfa Aesar,

Shanghai, China. Lot: 10180463; 3-MP was purchased from J&K

Scientiﬁc Ltd., Beijing, China. Lot: LD50Q10; CBZ (J&K Scientiﬁc Ltd.,

Beijing, China. Lot: LLA0P07), STP (TCI, Shanghai, China. Lot:

YDNLC-CP) and VPA (J&K Scientiﬁc Ltd., Beijing, China. Lot:

LV10N57) were used as reference anticonvulsant drugs for

comparison.

5.1.24.31. 3-(2,4,5-Trimethoxyphenyl)propyl (E)-3-(3,4,5-

trimethoxyphenyl)acrylate (85). White solid. Yield: 71.2%. Mp

50.0e50.4 ^ꢁC. TLC: R_f¼ 0.58 (1:1 EtOAc/hexanes). HPLC (purity

98.5%): t_R¼ 7.19 min ¹H NMR (600 MHz, Chloroform-d)

d 7.58 (d,

J ¼ 15.9 Hz, 1H), 6.76 (s, 2H), 6.71 (s, 1H), 6.51 (s, 1H), 6.36 (d,

J ¼ 15.9 Hz,1H), 4.22 (t, J ¼ 6.5 Hz, 2H), 3.89 (s, 6H), 3.88 (s, 3H), 3.86

(s, 3H), 3.83 (s, 3H), 3.80 (s, 3H), 2.68 (t, J ¼ 7.4 Hz, 2H), 1.98 (p,

J ¼ 6.7 Hz, 2H). ¹³C NMR (151 MHz, Chloroform-d)

d 167.14, 153.56,

151.69, 148.01, 144.68, 142.93, 140.18, 130.08, 121.19, 117.63, 114.42,

105.31, 97.86, 64.43, 61.11, 56.85, 56.39, 56.36, 56.29, 29.30, 26.42.

IR (KBr) 3448, 2942, 2839, 1704, 1634, 1582, 1522, 1510, 1461, 1423,

1397, 1339,1311, 1261, 1245,1229, 1229, 1207, 1154, 1129, 1065, 1037,

1004, 975, 854, 822 cm^ꢀ1. HRMS (þESI) 447.2015 [M þ H]^þ,

469.1833 [M

þ

Na]^þ(calcd. for C₂₄H₃₁O^þ₈447.2013 and

C

₂₄H₃₀O₈Na^þ469.1833).

5.1.25. General method for the preparation of cinnamic acid amide

derivatives (86 and 87)

A mixture of the compound 5 or 6 (5.0 mmol), cinnamic acid

derivatives (6 mmol), and HOBt (6.0 mmol) in DCM (15.0 mL) was

stirred for 10 min at room temperature. Successively, EDCI

(8.0 mmol) were added quickly in one portion. The reaction

mixture was continually stirred at room temperature for 10 h under

nitrogen and monitored by TLC. After workup and puriﬁcation as

same as the procedures of cinnamic acid esters, the ﬁnal amides

were obtained as solid substances in good yield.

5.2.2. Maximal electroshock seizure (MES) test

The MES test were carried out according to previously described

method [45]. In experiments with mice, a current of 50 mA with a

pulse frequency of 50 Hz for 0.2 s was applied via ear-clip elec-

trodes by an electronic stimulator (SEN-3201, Nihon Konden).

Protection against the spread of MES-induced seizures was deﬁned

as the absence of the tonic hind limb extension (hind limbs of an-

imals outstretched 180^ꢁto the plane of the body). For the vehicle

group, stimulation with this current caused 100% tonic hind limb

extension in mice. For the test groups, 0.5, 1, 2, 3 and 4 h after

administration of the compounds all the mice were treated with

current electrical stimulation. An effective dose protecting 50% of

the mice (ED₅₀) of the most active compounds against the tonic

hind limb extension and its associated 95% conﬁdence interval

were calculated by Probit analysis.

5.1.25.1. (E)-3-(3,4,5-trimethoxyphenyl)-N-((E)-3-(2,4,5-

trimethoxyphenyl)allyl) acrylamide (86). Light yellow solid. Yield:

80.8%. Mp 68.1e68.4 ^ꢁC. TLC: R_f¼ 0.30 (1:1 EtOAc/hexanes). HPLC

(purity 95.4%): t_R¼ 5.12 min ¹H NMR (500 MHz, Chloroform-d)

d

7.55 (d, J ¼ 15.5 Hz, 1H), 6.81 (s, 1H), 6.72 (s, 2H), 6.56 (s, 1H), 6.54

(d, J ¼ 9.2 Hz, 1H), 6.32 (d, J ¼ 15.5 Hz, 1H), 6.06 (ddd, J ¼ 5.1, 10.3,

17.1 Hz, 1H), 5.81 (ddt, J ¼ 1.6, 4.9, 8.6 Hz, 1H), 5.18e5.12 (m, 2H),

24

Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

5.2.3. scPentylenetetrazole (scPTZ)-induced seizure test

regression method in SPSS software.

The anticonvulsant activity of the test compounds was deter-

mined with scPTZ-induced seizure test. In this model of induced

seizure, 85 mg/kg of freshly prepared solution of PTZ was admin-

istered subcutaneously to all the mice. This produces tonic seizures

could last at least 5 s in 97% of animals tested. Each group of 4 mice

were tested at 0.25, 0.5, 1.0 and 2.0 h following doses of 100 and

300 mg/kg of compounds. The mice were considered to be pro-

tective when the compounds were in the absence of the effect of

PTZ on seizure threshold (last at least 5 s). The percentage of inci-

dence of tonic seizures, clonic seizures (jerking, twitching, etc.) as

well as the mortality rate were observed for 30 min after PTZ in-

jection. Additionally, the median effective dose (ED₅₀) and 95%

conﬁdence intervals of some most active compounds were calcu-

lated by Probit-regression method in SPSS software (Version 13.0).

5.2.7. LDH inhibition assay

5.2.7.1. UV-VIS spectrophotometry method. This assay was carried

out using, with slight modiﬁcation, the procedure described by

Carlotta [56] and Sada [12] respectively. The effect of compounds 55

to 87 on LDH enzyme activity was measured using puriﬁed human

lactate dehydrogenase isoforms 1 and 5 (Lee Biosolution, Inc. LDH1,

Lot No. MS7381; LDH5, Lot No: 04B2913). LDH activity was deter-

mined at 37 ^ꢁC by measuring the oxidation of NADH spectropho-

tometrically at various wavelengths (supporting Information

Table 4) in the direction of pyruvate to lactate. The apparent

Michaelis-Menten constants (K_m) of LDH1 and LDH5 for NADH and

pyruvate were measured from Lineweaver-Burk plots and deter-

mined in saturating conditions as described below. In 100 mM so-

dium phosphate buffer (pH 7.5), 0.015 units of LDH1 or LDH5 were

combined with 1.0 mM sodium pyruvate and 0.2 mM NADH. This

condition was used for the evaluation of the percent inhibition ratio

of the compounds. The reaction velocity of LDH1 or LDH5 was

determined spectrophotometrically (Agilent Cary 8453) by a

decrease in absorbance at the optimum detection wavelength

(ε ¼ 6.2 mM^ꢀ1cm^ꢀ1) of NADH, and the value of optical density was

determined at 30 s intervals for 4.5 min. K_mvalues for substrate

were determined from initial rate measurements at 37 ^ꢁC by

nonlinear regression analysis with the GraphPad Prism 5.0. Since all

of C series of compounds were poorly soluble in water, 0.5% DMSO

and 0.5% Tween-80 as cosolvent were added to reaction mixture

without reducing the LDH activity. In addition, inhibition mode and

K_ivalues for the representative compounds (65, 68, and 69) were

determined by the following conditions. In 100 mM sodium phos-

phate buffer (pH 7.5), 0.015 units of LDH1 or LDH5 were combined

with 0.2 mM NADH and 0.1 mMe1.0 mM sodium pyruvate. Then,

the inhibition mode and K_ivalues for each single inhibitor were

determined using Lineweaver-Burk plots.

5.2.4. 3-Mercaptopropionic acid (3-MP)-induced seizure test

The anticonvulsant activity of the selected compounds was

determined with sc3-MP-induced seizure test. In this model of

induced seizure, 60 mg/kg of freshly prepared solution of 3-MP was

administered subcutaneously to all the mice. It produced tonic

seizures that last at least 5 s in 97% of animals tested. Each group of

4 mice were tested at 0.25, 0.5, 1.0 and 2.0 h following doses of 100

and 300 mg/kg of each compound. The mice were considered

protected when the compound in the absence or presence of the

effect of 3-MP on seizure threshold (a 5 s episode of tonic convul-

sions). Meanwhile, the percentage of incidence of tonic seizures,

clonic seizures as well as the mortality rate were observed for

30 min after 3-MP injection. The median effective dose (ED₅₀) and

95% conﬁdence intervals of the most active compounds were

calculated by Probit-regression method in SPSS software.

5.2.5. Neurotoxicity screening

The neurotoxicity of compounds 55e87 was assessed by eval-

uating motor coordination (minimal motor impairment) in mice

using rotarod test. The mice were trained to position on a 4 cm

diameter rod that rotated at a constant speed of 24 rpm. Then,

trained mice were administrated ip. with test compounds at the

doses of 50, and 100 mg/kg 0.5, 1, and 2 h after compounds treat-

ment, each animal was placed on rotarod that rotated at 24 rpm.

Neurologic toxicity was deﬁned as the failure (drop more than 3

times from rod in 3 min) of the mice to remain on the rod for 3 min.

TD₅₀for the most active compounds (doses of 200, 400, 600, 800

and 1000 mg/kg, ip) at 0.5 h were also calculated based on Probit-

regression method in SPSS software.

5.2.7.2. Multiple reaction monitoring (MRM) method. The reaction

velocity of puriﬁed human LDH1 and LDH5 was determined by an

increase of lactic acid. The concentration of lactic acid was deter-

mined at 60 s intervals for 5 min. In the test, acetonitrile (100

was added to the portion of reaction mixture (100 L) to cease the

reaction, which was subjected to vortex (30 s), centrifugation

(9000 rpm), and water dilution (800 L) procedures. Then the

mL)

m

mixture was detected by MRM conditions (supporting

information).

5.2.8. Cells expressing GABAa receptors

5.2.6. Formalin-induced paw licking test

A stable HEK293T-GABAa cell line expressing GABAa receptor

Formalin-induced tonic pain were carried out according to

Hunskaar and Hole [77]. The selected compounds or vehicle were

administered by intraperitoneal injection to each mouse. Thirty

minutes after pretreatment, an intrapaw injection of 2.5% formalin

subunits

Ltd) was used for the study. Cells were maintained in cell culture

medium (DMEM, 11% FBS, 100 g/ml G418, 40 g/ml Hygromycin B,

80

g/ml Zeocin) and incubated at 37 ^ꢁC in a humidiﬁed incubator

a1b2g2 (kindly provided by PharmaCore Labs (PCL) Co.,

m

solution (25

m

L) was given to the right sub-plantar surface of the

with 5% CO₂and 95% air. 24e48 h prior to electrophysiological re-

cordings, cells were split and seeded onto round glass cover slips

placed in Petri dishes and maintained under the same incubation

and media conditions.

mice, and then the mice were returned to the observation cages.

The compound's efﬁcacy in the 2nd phase (15e30 min after

formalin injection) was viewed as predictive of efﬁcacy in animal

models of neuropathic pain [78]. Thus, in our study, the duration

time that the mice spent licking or biting its right hindpaw was

determined and recorded for each consecutive 5 min time binned

for 30 min after the injection of formalin in the 2nd-phase of the

test. The selected compound's main outcome measure was the 2nd-

phase value at which 50% of mice showed a paw lick duration of

nociceptive response less than 80 s (ED₅₀), which corresponds to a

50% reduction of the nociceptive response compared with the

mean of vehicle-treated (normal saline) mice. Then, the ED₅₀

values, with 95% conﬁdence intervals, were calculated using Probit-

5.2.9. Electrophysiological recordings

The electrophysiological characterization of stiripentol and

compound 70 at GABA_ARs was performed essentially as described

by Christian et al. [79]. Cover slips with cells were transferred to the

stage of a TI-S-FLU microscope (Nikon, Japan), and were perfused

with external solution at room temperature (22e25 ^ꢁC). The

external solution consisted of (in mM): 140 NaCl, 4.7 KCl, 1.0 MgCl₂,

2.0 CaCl₂, 11 glucose, and 10 HEPES (4-(2-hydroxyethyl)-1-

piperazineethanesul fonic acid) with pH ¼ 7.4 and osmolarity

Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

25

adjusted to 295e305 mOsm. Recording electrodes of 2e4 M

U

tip

ED₅₀

EDCI

effective dose (50%)

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

hydrochloride

hexamethylenetetramine

intraperitoneal

lithium aluminum hydride

lactate dehydrogenase

resistance were made with P97 micropipette puller (Sutter In-

strument Company, One Digital Drive, Novato, CA 94949) from

borosilicate glass tubings with ﬁlament (BF150-86-10, Sutter In-

strument Company) and ﬁlled with an internal solution of (in mM):

110 CsCl, 1.0 MgCl₂, 1.0 CaCl₂, 10 HEPES, 10 EGTA (Ethylene glycol-

bis(2-aminoethylether)- N,N,N⁰,N⁰-tetraacetic acid), 2.0 ATP-Na₂

and 25 TEA∙Cl with pH ¼ 7.2 and osmolarity adjusted to 300e310

mOsm. Patch-clamp techniques in voltage clamp mode were used

to record from HEK293T-GABAa cells in the whole-cell conﬁgura-

tion using an EPC-10 ampliﬁer (HEKA, Germany). Cells were held

at ꢀ50 mV during the recordings. Whole-cell currents were

analyzed using Patchmaster software (HEKA, Germany). To allow

quick switch and exchange of the test compounds, perfusion during

the recordings was achieved through an 8 Channel Bath Perfusion

System (VC³-8PG, ALA Scientiﬁc Instruments, Inc., USA). All ex-

periments were performed at room temperature.

HMTA

ip

LAH

LDH

NADH

S/N

ORTEP

PK

b-nicotinamide adenine dinucleotide disodium salt

Signal-to-Noise

oak ridge thermal-ellipsoid plot program

pharmacokinetics

structure-activity relationship

subcutaneous injection-pentylenetetrazole

3-mercaptopropionic acid

Traditional Chinese medicine

neurological impairment (toxicity, 50%)

triﬂuoroacetic acid

SAR

scPTZ

sc3-MP

TCM

TD₅₀

TFA

3,4,5-TMCA 3,4,5-trimethoxycinnamic acid

5.3. Pains

References

All of the active compounds were electronically ﬁltered for

structural attributes consistent with classiﬁcation as pan-assay

interference compounds (PAINS) and were found to be negative

[80,81].

[1] A. Vezzani, J. French, T. Bartfai, T.Z. Baram, The role of in ﬂ ammation in epi-

lepsy, Nat. Rev. Neurol. 7 (2011) 31e40.

[2] O. Devinsky, A. Vezzani, S. Najjar, N.C. De Lanerolle, M.A. Rogawski, Glia and

epilepsy: excitability and in ﬂ ammation, Trends Neurosci. 36 (2013) 174e184.

[3] D.C. Hesdorffer, G. Logroscino, E.K.T. Benn, N. Katri, G. Cascino, W.A. Hauser,

Notes

Estimating risk for developing epilepsy:

Rochester, Minnesota, Neurology 76 (2011) 23e27.

a population-based study in

[4] G. Seifert, G. Carmignoto, C. Steinhaeuser, Astrocyte dysfunction in epilepsy,

Brain Res. Rev. 63 (2010) 212e221.

The authors declare no competing ﬁnancial interest.

[5] R. Mohanraj, J. Norrie, L.J. Stephen, K. Kelly, N. Hitiris, M.J. Brodie, Mortality in

adults with newly diagnosed and chronic epilepsy: a retrospective compar-

ative study, Lancet Neurol. 5 (2006) 481e487.

Acknowledgments

[6] N.D. Lawn, W.R. Bamlet, K. Radhakrishnan, P.C. O'Brien, E.L. So, Injuries due to

seizures in persons with epilepsy: a population-based study, Neurology 63

(2004) 1565e1570.

[7] J. McCagh, J.E. Fisk, G.A. Baker, Epilepsy, psychosocial and cognitive func-

tioning, Epilepsy Res. 86 (2009) 1e14.

We thank Professor Xifeng Zhai of Xi'an Medical University for

help in molecular docking study and Dr. Wei Sun of Northwest

University for NMR analysis. This work was supported by The

Development and Application of Supercritical Fluid Chromatog-

raphy (2013YQ170525; subproject: Application Research of Super-

critical Fluid Chromatography in Chinese Traditional Medicine and

Its Metabolites, 2013YQ17052509), Program for Changjiang

Scholars and Innovative Research Team in University of Ministry of

Education of China (IRT_15R55), National Natural Science Founda-

tion of China (20875074), CAMS Initiative for Innovative Medicine

(2016-I2M-3e010), The Seventh Group of Hundred-Talent Program

of Shaanxi Province (2015), and The Project of Key Research and

Development Plan of Shaanxi (2017ZDCXL-SF-01-02-01).

[8] H.E. Scharfman, Metabolic control of epilepsy, Science 347 (2015) 1312e1313.

[9] U. Heinemann, D. Kaufer, A. Friedman, Blood-brain barrier dysfunction, TGF

b

signaling, and astrocyte dysfunction in epilepsy, Glia 60 (2012) 1251e1257.

[10] D.A. Coulter, T. Eid, Astrocytic regulation of glutamate homeostasis in epi-

lepsy, Glia 60 (2012) 1215e1226.

[11] R.M. Kaminski, M.A. Rogawski, H. Klitgaard, The potential of antiseizure drugs

and agents that act on novel molecular targets as antiepileptogenic treat-

ments, Neurotherapeutics 11 (2014) 385e400.

[12] N. Sada, S. Lee, T. Katsu, T. Otsuki, T. Inoue, Targeting LDH enzymes with a

stiripentol analog to treat epilepsy, Science 347 (2015) 1362e1367.

[13] M.K. Trojnar, K. Wojtal, M.P. Trojnar, S.J. Czuczwar, Stiripentol. A novel anti-

epileptic drug, Pharmacol. Rep. 57 (2005) 154e160.

[14] S. Auvin, C. Lecointe, N. Dupuis, B. Desnous, S. Lebon, P. Gressens, P. Dournaud,

Stiripentol exhibits higher anticonvulsant properties in the immature than in

the mature rat brain, Epilepsia 54 (2013) 2082e2090.

Appendix A. Supplementary data

[15] K. Eschbach, K.G. Knupp, Stiripentol for the treatment of seizures in Dravet

syndrome, Expert Rev. Clin. Pharmacol. 12 (2019) 379e388.

[16] X. He, Y. Bai, M. Zeng, Z. Zhao, Q. Zhang, N. Xu, F. Qin, X. Wei, M. Zhao, N. Wu,

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2019.1 11650.

Z. Li, Y. Zhang, T.-P. Fan, X. Zheng, Anticonvulsant activities of

a

-asaronol ((E)-

3'-hydroxyasarone), an active constituent derived from

a -asarone, Pharmacol.

Rep. 70 (2018) 69e74.

[17] A.T. Cartus, D. Schrenk, Metabolism of the carcinogen alpha-asarone in liver

microsomes, Food Chem. Toxicol. 87 (2016) 103e112.

Abbreviations used

[18] S. Haupenthal, K. Berg, M. Gruendken, S. Vallicotti, M. Hemgesberg, K. Sak,

D. Schrenk, M. Esselen, In vitro genotoxicity of carcinogenic asarone isomers,

Food Funct 8 (2017) 1227e1234.

ADME

AED

CBZ

Absorption, Distribution, Metabolism, Excretion

antiepileptic drug

carbamazepine

[19] L. Jimenez, G. Chamorro, M. Salazar, N. Pages, Teratological evaluation of a -

asarone in rats, Ann. Pharm. Fr. 46 (1988) 179e183.

CFDA

China Food and Drug Administration

[20] M. Salazar, S. Salazar, V. Ulloa, T. Mendoza, N. Pages, G. Chamoro, Teratogenic

effect of -asarone in mice, J. Toxicol. Clin. Exp. 12 (1992) 149e154.

m-CPBA m-chloroperbenzoic acid

CTCMMC Combination of traditional Chinese medicine molecular

chemistry

a

[21] P. Morales-Ramirez, E. Madrigal-Bujaidar, J. Mercader-Martinez, M. Cassani,

G. Gonzalez, G. Chamorro-Cevallos, M. Salazar-Jacobo, Sister-chromatid ex-

change induction produced by in vivo and in vitro exposure to alpha-asarone,

Mutat. Res., Genet. Toxicol. Test. 279 (1992) 269e273.

[22] S. Stegmueller, D. Schrenk, A.T. Cartus, Formation and fate of DNA adducts of

alpha- and beta-asarone in rat hepatocytes, Food Chem. Toxicol. 116 (2018)

138e146.

[23] C.-Y. Chen, X.-D. Wei, C.-R. Chen, 3,4,5-Trimethoxycinnamic acid, one of the

constituents of Polygalae Radix exerts anti-seizure effects by modulating

GABAAergic systems in mice, J. Pharmacol. Sci. 131 (2016) 1e5.

DIAD

diisopropyl azodicarboxylate

DIBAL-H diisobutyl aluminum hydride

DMAP

ED

EW

EC₅₀

4-dimethylaminopyridine

electron-donating

electron-withdrawing

effective concentration (50%)

26

Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

[24] J. Jiang, T. Ganesh, Y. Du, Y. Quan, G. Serrano, M. Qui, I. Speigel, A. Rojas,

N. Lelutiu, R. Dingledine, Small molecule antagonist reveals seizure-induced

mediation of neuronal injury by prostaglandin E2 receptor subtype EP2,

Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 3149e3154.

[25] T. Ganesh, J. Jiang, M.-S. Yang, R. Dingledine, Lead optimization studies of

cinnamic amide EP2 antagonists, J. Med. Chem. 57 (2014) 4173e4184.

[26] X. Zheng, P. Jia, Y. Bai, Research strategy for combination of traditional Chinese

medicine molecular chemistry based on the traditional theory of “Jun-Chen-

Zuo-Shi, . J. Northwest Univ. Nat. Sci. Ed. 45 (2015) 405e412.

[27] B. Meunier, Hybrid molecules with a dual mode of action: dream or reality?

Acc. Chem. Res. 41 (2008) 69e77.

5274e5286.

[52] H. Ucar, K. Van derpoorten, S. Cacciaguerra, S. Spampinato, J.P. Stables,

P. Depovere, M. Isa, B. Masereel, J. Delarge, J.H. Poupaert, Synthesis and

anticonvulsant activity of 2(3H)-Benzoxazolone and 2(3H)-Benzothiazolone

derivatives. [Erratum to document cited in CA128:252827], J. Med. Chem. 41

(1998) 3102.

[53] M. Poisson, F. Huguet, A. Savattier, F. Bakri-Logeais, G. Narcisse, A new type of

anticonvulsant, stiripentol. Pharmacological pro ﬁ le and neurochemical study,

Arzneim.-Forsch. 34 (1984) 199e204.

[54] M.N. Aboul-Enein, A.A. El-Azzouny, M.I. Attia, Y.A. Maklad, K.M. Amin,

M. Abdel-Rehim, M.F. El-Behairy, Design and synthesis of novel stiripentol

analogues as potential anticonvulsants, Eur. J. Med. Chem. 47 (2012) 360e369.

[55] A.M. King, C. Salome, J. Dinsmore, E. Salome-Grosjean, M. De Ryck,

R. Kaminski, A. Valade, H. Kohn, Primary amino acid derivatives: compounds

with anticonvulsant and neuropathic pain protection activities, J. Med. Chem.

54 (2011) 4815e4830.

[56] C. Granchi, S. Roy, C. Giacomelli, M. Macchia, T. Tuccinardi, A. Martinelli,

M. Lanza, L. Betti, G. Giannaccini, A. Lucacchini, N. Funel, L.G. Leon,

E. Giovannetti, G.J. Peters, R. Palchaudhuri, E.C. Calvaresi, P.J. Hergenrother,

F. Minutolo, Discovery of N-Hydroxyindole-Based inhibitors of human lactate

dehydrogenase isoform a (LDH-A) as starvation agents against cancer cells,

J. Med. Chem. 54 (2011) 1599e1612.

[57] C. Chiron, M.C. Marchand, A. Tran, E. Rey, P. d'Athis, J. Vincent, O. Dulac,

G. Pons, Stiripentol in severe myoclonic epilepsy in infancy: a randomized

placebo-controlled syndrome-dedicated trial, Lancet 356 (2000) 1638e1642.

[58] M.A. Kane, A.E. Folias, C. Wang, J.L. Napoli, Quantitative pro ﬁ ling of endoge-

nous retinoic acid in vivo and in vitro by tandem mass spectrometry, Anal.

Chem. 80 (2008) 1702e1708.

[59] S. Hirose, Mutant GABA(A) receptor subunits in genetic (idiopathic) epilepsy,

Prog. Brain Res. 213 (2014) 55e85.

[60] J.-Q. Kang, W. Shen, M. Lee, M.J. Gallagher, R.L. MacDonald, Slow degradation

[28] J. Qiu, Traditional medicine: a culture in the balance, Nature 448 (2007)

126e128.

[29] T.-P. Fan, J.-C. Yeh, K.W. Leung, P.Y.K. Yue, R.N.S. Wong, Angiogenesis: from

plants to blood vessels, Trends Pharmacol. Sci. 27 (2006) 297e309.

[30] C.-n. Xia, H.-b. Li, F. Liu, W.-x. Hu, Synthesis of trans-caffeate analogues and

their bioactivities against HIV-1 integrase and cancer cell lines, Bioorg. Med.

Chem. Lett 18 (2008) 6553e6557.

[31] X. Zheng, Preparation of

WO2016082780.

a -asaryl Esters for Treating Mental Diseases, 2016.

[32] B.D. Dickson, N. Dittrich, D. Barker, Synthesis of 2,3-syn-diarylpent-4-

enamides via acyl-Claisen rearrangements of substituted cinnamyl morpho-

lines: application to the synthesis of magnosalicin, Tetrahedron Lett. 53

(2012) 4464e4468.

[33] G.V.S. Reddy, G.V. Rao, R.V.K. Subramanyam, D.S. Iyengar, A new novel and

practical one pot methodology for conversion of alcohols to amines, Synth.

Commun. 30 (2000) 2233e2237.

[34] B. Dursina, R. Reents, C. Delon, Y. Wu, M. Kulharia, M. Thutewohl,

A. Veligodsky, A. Kalinin, V. Evstifeev, D. Ciobanu, S.E. Szedlacsek,

H. Waldmann, R.S. Goody, K. Alexandrov, Identi ﬁ cation and speci ﬁ city

pro ﬁ ling of protein prenyltransferase inhibitors using new ﬂ uorescent phos-

phoisoprenoids, J. Am. Chem. Soc. 128 (2006) 2822e2835.

and aggregation in vitro of mutant GABAA receptor

g 2(Q351X) subunits

[35] E. Marsault, H.R. Hoveyda, M.L. Peterson, C. Saint-Louis, A. Landry, M. Vezina,

L. Ouellet, Z. Wang, M. Ramaseshan, S. Beaubien, K. Benakli, S. Beauchemin,

R. Deziel, T. Peeters, G.L. Fraser, Discovery of a new class of macrocyclic an-

tagonists to the human motilin receptor, J. Med. Chem. 49 (2006) 7190e7197.

[36] T. Jensen, H. Pedersen, B. Bang-Andersen, R. Madsen, M. Jørgensen, Palladium-

catalyzed aryl aminationeheck cyclization cascade: a one-ﬂ ask approach to 3-

substituted indoles, Angew. Chem. Int. Ed. 47 (2008) 888e890.

[37] B. Sathish Kumar, A. Kumar, J. Singh, M. Hasanain, A. Singh, K. Fatima,

D.K. Yadav, V. Shukla, S. Luqman, F. Khan, D. Chanda, J. Sarkar, R. Konwar,

A. Dwivedi, A.S. Negi, Synthesis of 2-alkoxy and 2-benzyloxy analogues of

estradiol as anti-breast cancer agents through microtubule stabilization, Eur. J.

Med. Chem. 86 (2014) 740e751.

[38] D. Maes, M.E. Riveiro, C. Shayo, C. Davio, S. Debenedetti, N. De Kimpe, Total

synthesis of naturally occurring 5,6,7- and 5,7,8-trioxygenated coumarins,

Tetrahedron 64 (2008) 4438e4443.

[39] F. Benington, R.D. Morin, L.C. Clark, Mescaline analogs. II. TETRA- and PENTA-

METHOXY –PHENETHYLAMINES, J. Org. Chem. 20 (1955) 102e108.

[40] M.D. Reeder, G.S.C. Srikanth, S.B. Jones, S.L. Castle, Synthesis of the core

structure of acutumine, Org. Lett. 7 (2005) 1089e1092.

associated with epilepsy, J. Neurosci. 30 (2010) 13895e13905.

[61] X. Huang, M. Tian, C.C. Hernandez, N. Hu, R.L. MacDonald, The GABRG2

nonsense mutation, Q40X, associated with Dravet syndrome activated NMD

and generated

a truncated subunit that was partially rescued by

aminoglycoside-induced stop codon read-through, Neurobiol. Dis. 48 (2012)

115e123.

[62] R.M. McKerman, P.J. Whiting, Which GABAA-receptor subtypes really occur in

the brain? Trends Neurosci. 19 (1996) 139e143.

[63] P.S. Dragovich, B.P. Fauber, L.B. Corson, C.Z. Ding, C. Eigenbrot, H. Ge,

A.M. Giannetti, T. Hunsaker, S. Labadie, Y. Liu, S. Malek, B. Pan, D. Peterson,

K. Pitts, H.E. Purkey, S. Sideris, M. Ultsch, E. VanderPorten, B. Wei, Q. Xu, I. Yen,

Q. Yue, H. Zhang, X. Zhang, Identi ﬁ cation of substituted 2-thio-6-oxo-1,6-

dihydropyrimidines as inhibitors of human lactate dehydrogenase, Bioorg.

Med. Chem. Lett 23 (2013) 3186e3194.

[64] C. Granchi, S. Bertini, M. Macchia, F. Minutolo, Inhibitors of lactate dehydro-

genase isoforms and their therapeutic potentials, Curr. Med. Chem. 17 (2010)

672e697.

[65] Z. Fu, S. Huang, W. Su, M. Hong, Pd-catalyzed dearboxylative Heck coupling

with dioxygen as the terminal oxidant, Org. Lett. 12 (2010) 4992e4995.

[66] M. Yoshida, Y. Maeyama, K. Shishido, A synthetic study on bauhinoxepin J:

construction of a dibenzo[b,f]oxepin ring system by a DDQ-promoted oxida-

tive dearomatization-cyclization approach, Heterocycles 80 (2010) 623e629.

[67] M. Moir, R.H. Thomson, New cinnamaldehyde from Patagonula americana,

Phytochemistry 12 (1973) 2501e2503.

[68] R.K. Quinn, Z.A. Konst, S.E. Michalak, Y. Schmidt, A.R. Szklarski, A.R. Flores,

S. Nam, D.A. Horne, C.D. Vanderwal, E.J. Alexanian, Site-selective aliphatic C-H

chlorination using N-chloroamides enables a synthesis of chlorolissoclimide,

J. Am. Chem. Soc. 138 (2016) 696e702.

[41] M.B. Andrus, E.L. Meredith, E.J. Hicken, B.L. Simmons, R.R. Glancey, W. Ma,

Total synthesis of (þ )-Geldanamycin and (-)-o-Quinogeldanamycin: asym-

metric glycolate aldol reactions and biological evaluation, J. Org. Chem. 68

(2003) 8162e8169.

[42] K.M. Schmid, L. Jensen, S.T. Phillips, A self-immolative spacer that enables

tunable controlled release of phenols under neutral conditions, J. Org. Chem.

77 (2012) 4363e4374.

[43] R.I. Duclos, M. Johnston, S.K. Vadivel, A. Makriyannis, S.T. Glaser, S.J. Gatley,

A

methodology for radiolabeling of the endocannabinoid 2-

arachidonoylglycerol (2-AG), J. Org. Chem. 76 (2011) 2049e2055.

[44] W. Loscher, Critical review of current animal models of seizures and epilepsy

used in the discovery and development of new antiepileptic drugs, Seizure 20

(2011) 359e368.

[45] N. Hen, M. Bialer, B. Yagen, Syntheses and evaluation of anticonvulsant ac-

tivity of novel branched alkyl carbamates, J. Med. Chem. 55 (2012)

2835e2845.

[46] P. Yogeeswari, J.V. Ragavendran, D. Sriram, Y. Nageswari, R. Kavya,

N. Sreevatsan, K. Vanitha, J. Stables, Discovery of 4-aminobutyric acid de-

rivatives possessing anticonvulsant and antinociceptive activities: a hybrid

pharmacophore approach, J. Med. Chem. 50 (2007) 2459e2467.

[47] A. Fernandez-Guasti, K. Larsson, C. Beyer, GABAergic control of masculine

sexual behavior, Pharmacol, Biochem. Behav. 24 (1986) 1065e1070.

[48] A. Tjolsen, O.G. Berge, S. Hunskaar, J.H. Rosland, K. Hole, The formalin test: an

evaluation of the method, Pain 51 (1992) 5e17.

[49] M. Costigan, J. Scholz, C.J. Woolf, Neuropathic pain: a maladaptive response of

the nervous system to damage, Annu. Rev. Neurosci. 32 (2009) 1e32.

[50] S.H. White, J.H. Woodhead, K.S.K. Wilcox, H.H.J. Kupferberg, H.H.H. Wolf,

R.H. Levy, R.H. Mattson, B.S. Meldrum, General Principles of Discovery and

Preclinical Development of Antiepileptic Drugs, Lippincott Williams & Wil-

kins, Philadelphia, 2002.

[69] A.T. Shulgin, The six trimethoxyphenylisopropylpropylamines trimethox-

yamphetamines, J. Med. Chem. 9 (1966) 445e446.

[70] J. Yang, L. Weng, H. Zheng, Alternative synthesis of 1,2,3,4-tetramethoxy-5-

methylbenzene. A key intermediate for preparing coenzyme Q homologs

and analogs, Synth. Commun. 36 (2006) 2401e2405.

[71] L. Syper, K. Kloc, Mz. xl, J. lochowski, Synthesis of ubiquinone and menaqui-

none analogues by oxidative demethylation of alkenylhydroquinone ethers

with argentic oxide or ceric ammonium nitrat, Tetrahedron 36 (1980)

123e129.

[72] B. Shen, D. Loef ﬂ er, G. Reischl, H.-J. Machulla, K.-P. Zeller, Nucleophilic sub-

stitution of nitro groups by [18F]ﬂ uoride in methoxy-substituted ortho-

nitrobenzaldehydes-A systematic study, J. Fluorine Chem. 130 (2009)

216e224.

[73] M. Al Rahim, A. Nakajima, D. Saigusa, N. Tetsu, Y. Maruyama, M. Shibuya,

H. Yamakoshi, Y. Tomioka, Y. Iwabuchi, Y. Ohizumi, T. Yamakuni, 4'-Deme-

thylnobiletin, a bioactive metabolite of nobiletin enhancing PKA/ERK/CREB

signaling, rescues learning impairment associated with NMDA receptor

antagonism via stimulation of the ERK cascade, Biochemistry 48 (2009)

7713e7721.

[74] A. Brossi, P.N. Sharma, K. Takahashi, J.F. Chiang, I.L. Karle, G. Seibert, Tetra-

methoprim and pentamethoprim: synthesis, antibacterial properties and x-

ray structures, Helv. Chim. Acta 66 (1983) 795e800.

[51] K. Kaminski, M. Zagaja, J.J. Luszczki, A. Rapacz, M. Andres-Mach, G. Latacz,

K. Kiec-Kononowicz, Design, synthesis, and anticonvulsant activity of new

hybrid compounds derived from 2-(2,5-Dioxopyrrolidin-1-yl)propanamides

and 2-(2,5-Dioxopyrrolidin-1-yl)butanamides, J. Med. Chem. 58 (2015)

[75] M. Deodhar, D.S. Black, N. Kumar, Acid catalyzed stereoselective rearrange-

ment and dimerization of ﬂ avenes: synthesis of dependensin, Tetrahedron 63

(2007) 5227e5235.

Y. Bai et al. / European Journal of Medicinal Chemistry 183 (2019) 111650

27

[76] Guidelines for Research Involving Recombinant DNA Molecules, National

Institute of Health, Bethesda, 2002.

M. Larsen, B. Ebert, B. Bang-Andersen, P. Krogsgaard-Larsen, B. Frolund, 5-

Substituted imidazole-4-acetic acid analogues: synthesis, modeling, and

[77] S. Hunskaar, K. Hole, The formalin test in mice: dissociation between in-

ﬂ ammatory and non-in ﬂ ammatory pain, Pain 30 (1987) 103e114.

[78] K.C.P. Vissers, F. Geenen, R. Biermans, T.F. Meert, Pharmacological correlation

between the formalin test and the neuropathic pain behavior in different

species with chronic constriction injury, Pharmacol., Biochem. Behav. 84

(2006) 479e486.

pharmacological characterization of a series of novel

receptor agonists, J. Med. Chem. 50 (2007) 4147e4161.

g -aminobutyric AcidC

[80] J.B. Baell, G.A. Holloway, New substructure ﬁ lters for removal of Pan assay

interference compounds (PAINS) from screening libraries and for their

exclusion in bioassays, J. Med. Chem. 53 (2010) 2719e2740.

[81] J. Baell, M.A. Walters, Chemistry: chemical con artists foil drug discovery,

Nature 513 (2014) 481e483.

[79] C. Madsen, A.A. Jensen, T. Liljefors, U. Kristiansen, B. Nielsen, C.P. Hansen,

Article Doi

Polygala tenuifolia-Acori tatarinowii herbal pair as an inspiration for substituted cinnamic α-asaronol esters: Design, synthesis, anticonvulsant activity, and inhibition of lactate dehydrogenase study

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